Evaluation of a DNA microarray, the check-points ESBL/KPC array, for rapid detection of TEM, SHV, and CTX-M extended-spectrum beta-lactamases and KPC carbapenemases

Abstract

Extended-spectrum beta-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ss-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ss-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ss-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.

FIG. 1.

Principle of the Check-Points ESBL/KPC array. (A) When properly hybridized to a target sequence, the single-stranded nick lying between two adjacent probe arms is ligated. (B) Critical mismatches in the target sequence will cause ligation to fail, leaving the probe ends apart. (C) Successful ligation products are amplified by PCR using a single pair of amplimers annealing to complementary sequences included in the probes (gray and black boxes). (D) Unique ZIP codes (hashed box) assigned to each probe will be specifically captured by complementary oligonucleotides (cZIP codes, dotted box) spotted on the microarray. (E) Detection occurs by a biotin label incorporated at the 5′ end of one of the PCR primers. The system can be multiplexed with many different probes, each bearing a unique ZIP code. The successive reactions are processed in a single tube. S, substrate; P, product.

FIG. 2.

Typical DNA microarray pictures obtained with the Check-Points ESBL/KPC array setup. This format uses a DNA microarray fixed at the bottom of a microreaction vial. The microarray consists of unique complementary (cZIP) oligonucleotides targeting individual probes. When hybridization of the PCR-amplified ligation products to the microarray is complete, colorimetric detection of the positive reactions is initiated. Polygons delineate panels in the array. Each panel defines the typing results of one strain and consists of control spots and specific marker spots, which are numbered from 1 to 96. (A) Theoretical display of the array probes for strain 1 (panel I, defined by a thin border), strain 2 (panel II, defined by a medium-thickness border) and strain 3 (panel III, defined by a thick border). (B) Array results for E. coli CFVL (KPC-2, TEM-1, CTX-M-9; panel I), K. pneumoniae KPS2 (SHV-1; panel II), and Providencia stuartii PS (panel III). (C) Array results for K. pneumoniae H1521-6 (KPC-2 TEM-1, CTX-M-15, SHV-1; panel I), K. pneumoniae KN2303 (KPC-2, SHV-5, SHV-11; panel II), and K. pneumoniae GR (KPC-2, TEM-1, SHV-12, SHV-11; panel III). (D) Array results for K. pneumoniae ILT-3 (CTX-M-19, TEM-1, SHV-1; panel I), Proteus mirabilis 18821 (TEM-1; panel II), and Enterobacter aerogenes BG08022153 (TEM-24; panel III).