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We evaluated the ability of a commercial microarray system (Check KPC/ESBL; Check-Points Health BV) to detect clinically important class A β-lactamase genes. A total of 106 Gram-negative strains were tested. The following sensitivity and specificity results were recorded, respectively: for blaSHV, 98.8% and 100%; for blaTEM, 100% and 96.4%; and for blaCTX-M and blaKPC, 100% and 100%.
The spread of class A or group 2be extended-spectrum β-lactamases (ESBLs) represents an emerging public-health concern (1, 17). Among the organisms of the Enterobacteriaceae family (e.g., Klebsiella pneumoniae and Escherichia coli), the most frequently detected and clinically important ESBLs belong to the TEM, SHV, and CTX-M families (17). While TEM- and SHV-type ESBLs arise via substitutions in strategically positioned amino acids (e.g., Gly238 and Arg164) from the natural narrow-spectrum TEM-1, TEM-2, or SHV-1 β-lactamase genes, all currently identified CTX-M enzymes demonstrate an ESBL phenotype (7, 14).
The ability to rapidly identify narrow-spectrum β-lactamases (e.g., SHV-11 and TEM-1) and ESBLs (e.g., SHV-5 and SHV-12 or TEM-10) has important clinical implications. Usually, Enterobacteriaceae species producing narrow-spectrum enzymes are resistant to penicillins and narrow-spectrum cephalosporins, whereas those producing ESBLs manifest resistance to extended-spectrum oxyimino-cephalosporins and aztreonam (14). Since resistance to quinolones and aminoglycosides is frequently observed among ESBL producers, carbapenems represent one of the therapeutic options of last resort for life-threatening infections due to these organisms (6, 14).
In some geographic areas, the spread of carbapenemases belonging to class A (e.g., KPCs), class B (e.g., VIMs and IMPs), and class D (e.g., OXA-48) has significantly compromised the clinical use of carbapenems, consigning clinicians to the use of “last-line” antimicrobials such as colistin (2, 19). In particular, the KPC β-lactamases (primarily KPC-2 and KPC-3) are the serine carbapenemases that are most widespread in the United States, and strains producing these enzymes are responsible for numerous outbreaks with high mortality rates (3, 9, 13). Although nine KPC-type β-lactamases have been described, their susceptibility profiles are similar, rendering the differentiation of these variants less clinically relevant (13, 22).
Prompt and appropriate antibiotic treatment for infections due to ESBLs- and/or KPC-producing Enterobacteriaceae may positively affect the final outcome for infected patients (6, 13). Unfortunately, standard and confirmatory phenotypic tests may fail to identify ESBL- and, more frequently, KPC-producing organisms. For the latter group, the use of the modified Hodge test delays the final report by an additional 24 h (11, 12, 21). Therefore, a rapid and reliable method is needed to perform a quick and accurate analysis of the most important bla genes possessed by clinical isolates.
Microarray technologies are promising genotyping systems that possess a high multiplexing capacity and can be used for detecting different β-lactamase genes that are present in a single strain (8, 10, 23). This ability can assist clinicians in directing antimicrobial therapy. In the present work, we evaluated the ability of Check KPC/ESBL (Check-Points Health BV, Wageningen, Netherlands), the first rapid, commercially available, microarray-based diagnostic test system for detection and identification of bla genes belonging to the TEM, SHV, CTX-M, and KPC types. This system can detect single nucleotide polymorphisms found in the most important TEM- and SHV-type ESBLs (www.lahey.org/studies), including single mutations corresponding to amino acid positions Val84Ile, Glu104Lys, Arg164Ser/His/Cys, and Gly238Ser in TEMs and Gly238Ser/Ala and/or Glu240Lys in SHVs (7).
A total of 102 Enterobacteriaceae and four Acinetobacter baumannii isolates possessing different bla genes were tested (Table (Table1;1; see also Table S1 in the supplemental material). The majority of strains (n = 61) had previously been characterized (3-5, 15), whereas the bla genes of the remaining isolates were characterized by PCR amplification, standard DNA sequencing, and analytical isoelectric focusing (aIEF) as previously described (4). In this collection, isolates possessed an average of three different bla genes (range, one to five; see Table S1 in the supplemental material). The collection also included K. pneumoniae ATCC 700603, which produces the SHV-18 ESBL (20), and six E. coli DH10B control strains in which single bla genes are carried in different plasmid vectors (see Table S1 in the supplemental material).
Genomic DNA of strains was extracted from overnight colonies grown on blood agar (BBL, Sparks, MD) by the use of a DNeasy blood and tissue kit (Qiagen Sciences, Germantown, MD). Microarray assays were performed according to the instructions of the manufacturer (Check-Points Health BV). Briefly, templates of the target bla DNA sequences are generated during the ligation step. These templates are then amplified, and the products are hybridized in specific array tubes. Tubes are then inserted in the array tube reader upon completion of the detection reaction, and images are acquired and interpreted with software supplied by the manufacturer (Fig. (Fig.1).1). For 50 isolates, the complete procedure (i.e., from genomic DNA extraction to results) can be performed in approximately 8 h.
Overall, the Check KPC/ESBL system correctly identified representatives of the four bla gene families tested, including differentiation between non-ESBL and ESBL genes, in 97 of 106 isolates (91.5%). Specificities of 100% were recorded for the blaKPC, blaSHV, and blaCTX-M genes, whereas one false positive was reported for blaTEM genes (specificity of 96.4%). The system detected all blaKPC-, blaTEM-, and blaCTX-M-possessing isolates, including differentiation of ESBL from non-ESBL blaTEM-containing strains (Table (Table1).1). Notably, all blaCTX-M genes detected were classified into the appropriate family group (i.e., group I, CTX-M-1-like; group II, CTX-M-2-like; group III, CTX-M-8-like; group IV, CTX-M-9-like; group V, CTX-M-25/CTX-M-26) according to the classification method of Pitout et al. (16) (see Table S1 in the supplemental material).
Detection and recognition of the blaSHV genes showed 92.5% agreement, with sensitivity and specificity of 98.8% and 100%, respectively (Table (Table1).1). Only 1 in 44 blaESBL-positive strains (i.e., blaSHV-38-positive strains) was not identified (97.7% agreement). SHV-38 is a very rare chromosomal ESBL enzyme (group 2be) that was found in a single clinical isolate. It possesses a unique amino acid substitution (i.e., Ala146Val) and is capable of conferring resistance to ceftazidime and imipenem (18). The amino acid at position 146 is not included in those analyzed by the Check KPC/ESBL system.
Six strains with non-ESBL blaSHV genes were misclassified as ESBLs (Table (Table2).2). Notably, three of these were blaSHV-11-positive K. pneumoniae isolates (non-ESBL), which showed β-lactamase bands at pIs of 7.6 and 8.2 by aIEF and double spikes at positions 238 and/or 240 in the DNA sequencing traces of the blaSHV gene. This pattern is consistent with the possible production of an SHV-ESBL (along with the non-ESBL SHV-11) that was not detected with a cloning and DNA sequencing method that we previously employed (4). Therefore, blaSHV-positive total agreement and the overall agreement (i.e., all bla genes correctly reported) would improve by 2.8% if these three strains were classified as ESBL producers (Table (Table11).
The data presented above also support the previous observation that standard DNA sequencing of PCR amplification products fails to accurately detect more than one bla gene of a given family (4). In particular, many K. pneumoniae isolates possessing both blaSHV-11 (non-ESBL) and blaSHV-12 (ESBL) genes were initially identified incorrectly as blaSHV-11-positive isolates only with standard DNA sequence analysis (4). In contrast, the microarray can accurately identify the blaESBL gene (e.g., blaSHV-12) regardless of the coexistence of additional blanon-ESBL genes (e.g., blaSHV-1 and/or blaSHV-11) (see Table S1 in the supplemental material).
In conclusion, the results of the present work show that the microarray Check KPC/ESBL system is a highly accurate tool for detection of the clinically important β-lactamase genes found among contemporary Gram-negative organisms. Due to its rapid performance, this platform could be used in epidemiological or infection control studies in which large collections of isolates need to be characterized. Furthermore, the use of Check KPC/ESBL in clinical practice may lead to more appropriate use of antimicrobial agents, reduction of costs, and improved patient outcomes. More-extensive evaluations (e.g., using clinical isolates possessing bla genes not tested in this study) are needed to establish the full potential of this methodology for detecting different resistance genes.
This work was supported in part by the Veterans Affairs Merit Review Program (R.A.B.), the National Institutes of Health (grant RO3-AI081036 to R.A.B.), and the Geriatric Research Education and Clinical Center (grant VISN 10 to R.A.B.).
We thank Sarah Drawz for the critical revision of the manuscript and Francesco Luzzaro, Antonio Q. Toniolo, John Quale, David L. Paterson, Gerri S. Hall, and Stephen G. Jenkins for providing clinical isolates. We also thank Check-Points for the technical support and for providing the material necessary for the study.
Published ahead of print on 26 May 2010.
†Supplemental material for this article may be found at http://jcm.asm.org/.