This work is a report on the development of a method of hybridization analysis on DNA microarrays for the simultaneous identification and typing of carbapenemase-encoding genes. These enzymes are produced by the microorganisms that are responsible for causing infectious diseases. The method involves several steps, including DNA extraction from clinical samples and amplification of carbapenemase genes by multiplex PCR with simultaneous labelling by biotin. Following that, hybridization of the labeled PCR products with oligonucleotide probes immobilized on the surface of a nitrocellulose-based DNA microarray occurs. The biotin molecules attached to the DNA duplexes are detected by using conjugates of streptavidin-horseradish peroxidase, which is then quantified by colorimetric detection of the enzyme. We have designed the required oligonucleotide probes and optimized the conditions of the membrane microarray-based hybridization analysis. Our method allows to identify 7 types of carbapenemase genes belonging to the molecular classes A, B, and D, and it also allows additional typing into genetic subgroups. The microarrays have been tested with the control strains producing the carbapenemase genes which have been characterized by sequencing. The developed method of hybridization analysis was employed to investigate clinical strains ofPseudomonasspp. andAcinetobacterspp., which produce carbapenemases of different classes based on phenotypic testing. All strains ofAcinetobacter baumaniiresistant to carbapenems were producers of two carbapenemase OXA-type genes (OXA-51, in combination with OXA-23 (1 strain), OXA-40 (5 strains), or OXA-58 (4 strains)). The metallo-β-lactamase VIM-2 type gene was detected in allPseudomonas aeruginosastrains resistant to carbapenems. Testing of carbapenem-sensitive strains did not detect any carbapenemase genes. The microarray method for the identification of carbapenemase genes is very accurate and highly productive. It can be employed in clinical microbiological laboratories for the identification and study of carbapenemase epidemiology.
Keywords: DNA microarrays; antibiotic resistance; carbapenemases; colorimetric detection; horseradish peroxidase.