Using Nucleic Acid Microarrays To Perform Molecular Epidemiology and Detect Novel β-Lactamases: a Snapshot of Extended-Spectrum β-Lactamases throughout the World

Abstract

The worldwide dissemination of extended-spectrum-␤-lactamase (ESBL)-and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n ‫؍‬ 1,093) and ESBL-negative isolates (n ‫؍‬ 59), obtained in 2008 -2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla ESBL (bla SHV , bla TEM , and bla CTX-M genes of groups 1, 2, 9, and 8/25) and bla KPC genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla ESBL and bla KPC genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla ESBL gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla TEM (n ‫؍‬ 5), bla SHV (n ‫؍‬ 11), bla CTX-M (n ‫؍‬ 19), and bla KPC (n ‫؍‬ 3) were detected. A new bla SHV variant, bla SHV-129 , and a new bla KPC variant, bla KPC-11 , were also identified. The most common bla genes found in this study were bla CTX-M-15 , bla CTX-M-14 , and bla SHV-12 . Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla ESBL genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new ␤-lactamase variants bla SHV-129 and bla KPC-11 .

Evaluation of the Check-Points Check MDR CT103 and CT103 XL Microarray Kits by Use of Preparatory Rapid Cell Lysis

“…Given this, a comprehensive approach to detect resistance genes may be appropriate for MDRE. Studies employing microarray-based molecular tests that target a large number of resistance genes suggest that such platforms may serve this role (5,(12)(13)(14)(15). Cuzon et al evaluated the Check-Points Check MDR CT103 (Check-Points Health B.V., Wageningen, The Netherlands) microarray kit by using 187 clinical isolates and reported excellent specificity (16).…”

“…cols et al, who found the Check-KPC ESBL microarray to exhibit incomplete sensitivity for these targets (13). Compared to the historical characterization of the study isolates, the new testing identified an additional 49 resistance genes (34 bla TEM , 7 bla SHV , 3 bla CMY-II , 3 bla CTX-M-9 group genes, and 2 bla CTX-M-1 group genes) in 45 isolates (46 and 48 targets detected by CT103 and CT103 XL, respectively), all of which were confirmed with conventional PCR, highlighting the added utility of microarray testing (Table 1).…”

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

“…The commercially available microarray assays from Check-Points enable only the identification of genes and mutation hot spots relevant to resistance caused by ESBLs and carbapenemases, including the detection of bla KPC (4,5,7,9,22,23,44,47). However, the Check-Points system can be used as a reliable screening tool to guide PCR sequencing, allowing in this way an identification of single variants (14). The capability to identify single variants of the KPC gene might not have been a requirement in the past, as there were only a very limited number of KPC variants reported showing very similar phenotypes.…”

“…To identify all the different variants, sequencing is the gold standard, but this method is very time consuming and too demanding for routine clinical diagnostics. An alternative method is the use of a DNA microarray, which allows for rapid identification of SNPs and parallel detection of several resistance genes (4,5,7,14,15,22,23,44,47). However, the currently described methods for KPC gene detection (the Check-MDR CT101, CT102, and CT103 assays and Check-KPC extended-spectrum beta-lactamase [ESBL] microarray [Check-Points Health BV, Wageningen, Netherlands] and the hyplex SuperBug ID test system [Amplex BioSystems GmbH, Gießen, Germany]) do not allow for differentiation between the KPC variants.…”

Conventional Antibiotics – Revitalized by New Agents

“…The classification of bacterial beta-lactamases is complicated. We have used the Bush (2013) system in this paper, bearing in mind that Extended Spectrum BetaLactamases (ESBLs which include TEM and SHV) and carbapenemases(such as NDM and KPC) in Gram-negatives are thought to be of the greatest clinical importance because they are difficult to treat and are relatively common in many countries [33,34]. Beta-lactamases can be Pfizer, Philadelphia, PA, USA).…”