Laboratory and Clinical Evaluation of DNA Microarray for the Detection of Carbapenemase Genes in Gram-Negative Bacteria from Hospitalized Patients

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

Rapid Detection and Analysis of Raman Spectra of Bacteria in Multiple Fields of View Based on Image Stitching Technique

Background: Due to antibiotic abuse, the problem of bacterial resistance is becoming increasingly serious, and rapid detection of bacterial resistance has become an urgent issue. Because under the action of antibiotics, different active bacteria have different metabolism of heavy water, antibiotic resistance of bacteria can be identified according to the existence of a C-D peak in the 2030–2400 cm-1 range in the Raman spectrum. Methods: To ensure data veracity, a large number of bacteria need to be detected, however, due to the limitation of the field of view of the high magnification objective, the number of single cells in a single field of view is very small. By combining an image stitching algorithm, image recognition algorithm, and processing of Raman spectrum and peak-seeking algorithm, can identify and locate single cells in multiple fields of view at one time and can discriminate whether they are Antimicrobial-resistant bacteria. Results: In experiments 1 and 2, 2706 bacteria in 9 × 11 fields of view and 2048 bacteria in 11 × 11 fields of view were detected. Results showed that in experiment 1, there are 1137 antibiotic-resistant bacteria, accounting for 42%, and 1569 sensitive bacteria, accounting for 58%. In experiment 2, there are 1087 antibiotic-resistant bacteria, accounting for 53%, and 961 sensitive bacteria, accounting for 47%. It showed excellent performance in terms of speed and recognition accuracy as compared to traditional manual detection approaches. And solves the problems of low accuracy of data, a large number of manual experiments, and low efficiency due to the small number of single cells in the high magnification field of view and different peak-seeking parameters of different Raman spectra. Conclusions: The detection and analysis method of bacterial Raman spectra based on image stitching can be used for unattended, automatic, rapid and accurate detection of single cells at high magnification with multiple fields of view. With the characteristics of automatic, high-throughput, rapid, and accurate identification, it can be used as an unattended, universal and non-invasive means to measure antibiotic-resistant bacteria to screen for effective antibiotics, which is of great importance for studying the persistence and spread of antibiotics in bacterial pathogens

Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients

Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world’s population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19⁎2 and CYP2C19⁎3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2 ng/μL. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori