Dataset Information

Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

ABSTRACT: Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.

SUBMITTER: Braun SD

PROVIDER: S-EPMC4113224 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

Braun Sascha D SD Monecke Stefan S Thürmer Alexander A Ruppelt Antje A Makarewicz Oliwia O Pletz Mathias M Reiβig Annett A Slickers Peter P Ehricht Ralf R

PloS one 20140728 7

Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and n ...[more]

PMID: 25068267

Similar Datasets

Project description:BackgroundThe prevalence of a variety of carbapenemases in Gram-negative bacteria (GNB) has posed a global threat on clinical control and management. Monitoring and controlling the carbapenemase-producing GNB became imperative tasks for many healthcare centers. The aim of this study was to develop a high-throughput, specific, sensitive, and rapid DNA microarray-based method for the diagnosis, phenotypic confirmation, and molecular epidemiological study of carbapenemase genes.MethodsWe targeted a panel of eight carbapenemase genes, including bla KPC, bla NDM-1, bla OXA-23, bla OXA-48, bla OXA-51, bla IMP, bla VIM, and bla DIM for detection. Ultrasensitive chemiluminescence (CL) detection method was developed and used to simultaneously detect eight carbapenemase genes, and plasmids were established as positive or limit of detection (LOD) reference materials. Antibiotic susceptibility was determined by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines in order to screen clinical isolates resistant to carbapenem antibiotics as well as Sanger sequencing which was used to confirm the reliability of the results presented by DNA microarray.ResultsEight carbapenemase genes could be detected with high sensitivity and specificity. The absolute LOD of this strategy to detect serially diluted plasmids of eight carbapenemase genes was 102- 103copies/μL. Then, 416 specimens collected from hospital were detected and the results showed 96.6% concordance between the phenotypic and microarray tests. Compared with Sanger sequencing, a specificity and sensitivity of 100% were recorded for bla NDM-1, bla IMP, bla VIM, and bla DIM genes. The specificity for bla KPC, bla OXA-23, bla OXA-48, and bla OXA-51 genes was 100% and the sensitivity was 98.5%, 97.6%, 95.7%, and 97.9%, respectively. The overall consistency rate between the sequencing and microarray is 97.8%.ConclusionsThe proposed ultrasensitive CL imaging DNA hybridization has high specificity, sensitivity, and reproducibility and could detect and differentiate clinical specimens that carried various carbapenemase genes, suggesting that the method can conveniently be customized for high-throughput detection of the carbapenemase-producing GNB and can be easily adapted for various clinical applications.

Dataset Information

Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

Publications

Rapid identification of carbapenemase genes in gram-negative bacteria with an oligonucleotide microarray-based assay.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets