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.

The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside resistance genes in urinary tract infections in Saudi Arabia

“…For the microarray DNA analysis (Alere Technologies, Jena, Germany) [15], the ESBL/AmpC/carbapenemase genes were evaluated based on the manufacturer’s protocols ( bla ACC , bla ACT , bla CMY , bla KHM , bla MOX - CMY9 , bla CTX - M - 1 , bla CTX - M - 15 , bla CTX - M 2 , bla CTX - M - 8 , bla CTX - M - 9 , bla CTX - M - 26 , bla DHA - 1 , bla FOX , bla GES - 1 , bla G1M - 1 , bla MI - 3 , bla IMP , bla KPC , bla LAP - 1 , bla LEN - 1 , bla MOX , bla OXA - 1 , bla OXA - 2 , bla OXA - 7 , bla OXA - 9 , bla OXA - 23 , bla OXA - 23 , bla OXA - 40 , bla OXA - 48 , bla OXA - 51 , bla OXA - 54 , bla OXA - 55 , bla OXA - 58 , bla OXA - 60 , bla PER1 , bla PER2 , bla PSE - 1 , bla SHF - 1 , bla SHV , bla SEM - 1 , bla SPM - 1 , bla TEM , bla VEB - 1 , and bla VIM ). For the PCR analysis, DNA templates were obtained by boiling 500 µL of bacterial cells (OD 600  = 1.0) in a sterile single 1.5 mL Eppendorf tube in a water bath at 100 °C.…”

“…Molecular genotyping has been used concurrently with phenotyping techniques to detect and confirm antimicrobial drug resistance data and to detect Gram-negative ESBL producing bacteria [10]. PCR, multiplex PCR and oligonucleotide microarray-based assays have been developed and used to monitor the emergence of ESBLs and many other drug-resistant genes from E. coli , K. pneumonia and A. baumannii [11–15]. Strain characterization by multilocus sequence typing (MLST) is the method of choice in many clinical and research laboratories due to its high discriminatory power [16, 17].…”

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria

“…baumannii strains to identify the species, carbapenemases, ESBLs and narrow spectrum β ‐lactamases genes in a single reaction, with 98·2 and 97·4% sensitivity and specificity respectively (Braun et al . ). This commercial microarray provides a report on the species and resistance genes after analysing signals picked from hybridized probes on an Array Mate Reader (Braun et al .…”

“…This commercial microarray provides a report on the species and resistance genes after analysing signals picked from hybridized probes on an Array Mate Reader (Braun et al . ). In this evaluation, an OXA‐23 and OXA‐48 genes were not detected, and two K. oxytoca and one Kl.…”

The Changing Role of the Clinical Microbiology Laboratory in Defining Resistance in Gram-negatives

“…113,[122][123][124] This platform was evaluated against a panel of 117 Gram-negative isolates, showing an overall 100% concordance rate with the PCR method. 125 Nanosphere Technology (Northbrook, IL) has developed a promising automated microarray (Verigene Nucleic Acid Test) for detecting bacterial species and resistance genes within 2 hours. 126,127 In particular, the Verigene Gram-negative Blood Culture Nucleic Acid Test is able to identify the most frequent Gram-negative species along with CTX-M, KPC, NDM, VIM, IMP, and OXA enzymes.…”