Abstract
Background The 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. Methods We targeted a panel of eight carbapenemase genes, including blaKPC, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-51, blaIMP, blaVIM, and blaDIM 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. Results Eight 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 blaNDM-1, blaIMP, blaVIM, and blaDIM genes. The specificity for blaKPC, blaOXA-23, blaOXA-48, and blaOXA-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%. Conclusions The 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.
Highlights
Carbapenems are a class of β-lactam antibiotics with a broad spectrum and served as the last line against ESBLs efficiently and stably [1]
To determine the threshold value for differentiating positive and negative microarray signal intensities, we have performed pilot microarray hybridization experiments using the Grampositive bacterial strain S. aureus 04018 as negative controls and carbapenemase plasmids of 3×103 copies/μL as positive controls under the conditions specified in Materials and methods
We developed a novel microarray method to detect carbapenemase genes that could be applied to clinical diagnosis and identification of carbapenemase-producing Gram-negative bacteria (GNB)
Summary
Carbapenems are a class of β-lactam antibiotics with a broad spectrum and served as the last line against ESBLs efficiently and stably [1]. Mass spectrometry cannot provide molecular epidemiological information and is cost-ineffective Addressing these deficiencies, we propose a novel DNA microarraybased method for rapid, sensitive, and specific detection of clinical carbapenamase-producing samples. 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. The 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 highthroughput detection of the carbapenemase-producing GNB and can be adapted for various clinical applications
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