T. Naas,* G. Cuzon, H. Truong, S. Bernabeu, and P. Nordmann

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2010, p Vol. 54, No /10/$12.00 doi: /aac Copyright 2010, American Society for Microbiology. All Rights Reserved. Evaluation of a DNA Microarray, the Check-Points ESBL/KPC Array, for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum -Lactamases and KPC Carbapenemases T. Naas,* G. Cuzon, H. Truong, S. Bernabeu, and P. Nordmann Service de Bactériologie-Virologie, INSERM U914: Emerging Resistance to Antibiotics, Hôpital de Bicêtre, Le Kremlin- Bicêtre, and Assistance Publique-Hôpitaux de Paris, Faculté demédecine Paris-Sud, Paris, France Received 11 September 2009/Returned for modification 13 December 2009/Accepted 1 June 2010 Extended-spectrum ß-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. * Corresponding author. Mailing address: Service de Bactériologie- Virologie, INSERM U914: Emerging Resistance to Antibiotics, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France. Phone: Fax: thierry.naas@bct.ap-hop-paris.fr. Published ahead of print on 14 June Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemase (KPC) are reported increasingly in Gram-negative bacilli (GNB) (5, 6, 17, 18, 25, 30). KPC producers, initially identified in the United States, are now reported worldwide, and illnesses caused by them have become endemic in some regions (25). Isolates expressing KPC enzymes may be reported as susceptible to carbapenems due to heterogeneous and variable levels of expression of -lactam resistance. The vast majority of ESBLs belong to the TEM, SHV, and CTX-M types (5, 18, 28). These ß-lactamases are encoded by plasmid-located genes and therefore can very easily spread among Enterobacteriaceae (6, 14, 16). More than 160 TEMtype and 110 SHV-type ß-lactamases have been identified worldwide. Amino acid substitutions at many sites in TEM-1 ß-lactamases have been documented, but those at positions 104, 164, 238, and 240 most often lead to an ESBL phenotype (5, 28). As with TEM, SHV-type ESBLs have one or more amino acid substitutions located around the active site compared to SHV-1: substitutions at positions 238 and/or 240 are the most common and are associated with resistance to ceftazidime, cefotaxime, and aztreonam. Less commonly, an alteration at positions 146 or 179 provides ceftazidime resistance (28). Unlike TEM/SHV enzymes, all the CTX-M enzymes are ESBLs (6, 28). More than 80 CTX-M-variants, sharing 71 to 98% amino acid sequence identities, have now been described and are divided now into five groups (groups CTX-M-1, CTX- M-2, CTX-M-9, CTX-M-8, and CTX-M-25) based on amino acid sequence identity (5). Detection of ESBLs is primarily based on phenotypic testing, such as evidencing a synergy image using the double-disk synergy test performed with expanded-spectrum cephalosporins (ESC) and ticarcillin-clavulanic acid disks (3, 10, 23). This test is not always obvious and is usually time-consuming since it requires subculturing or the use of cloxacillin-containing plates to inhibit the naturally occurring and plasmid-mediated cephalosporinases. Unambiguous identification of KPCs by phenotypic methods is relatively difficult (25). Over the last 20 years, alternative strategies aimed at replacing or complementing traditional phenotypic methods have been proposed. Standard PCR and gene sequencing is still the most widely used technique. Other molecular detection techniques for ESBLs and KPC genes have been proposed, but none have been really suited for routine detection (1, 4, 8, 9, 11, 13, 15, 19, 20, 22, 24, 26, 27, 29, 31, 39), since usually only one ESBL/KPC gene is detected at a time. Finally, the presence of narrow-spectrum variants of TEM and SHV types may complicate significantly the molecular detection of TEM/SHV-type ESBLs (28). Microarray technology has recently been developed for the typing of Salmonella isolates (37, 38). This technology has the 3086

2 VOL. 54, 2010 MICROARRAY FOR TEM, SHV, CTX-M ESBL, AND KPC DETECTION 3087 TABLE 1. DNA array results on various clinical isolates harboring ß-lactamase genes Species a No. bla KPC bla CTX-M b bla SHV bla TEM Agreements g ESBL c Non-ESBL d ESBL e Non-ESBL f PCR h Array i % j PCR Array % PCR Array % PCR Array % PCR Array % PCR Array % Enterobacteria K. pneumoniae /62 89 E. coli / E. cloacae / S. marcescens /5 100 C. freundii /3 100 E. aerogenes /2 100 P. stuartii /1 100 P. mirabilis /4 100 K oxytoca /3 100 Nonfermenters P. aeruginosa / A. baumannii /4 100 Total k l m n / a Includes control isolates (producing VEB-1, PER-1, GES-7, ACC-1, DHA-2, OXA-1, OXA-18, OXA-23, OXA-48, IMP-1, VIM-1, and KOXY). b Includes CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14, CTX-M-15, and CTX-M-19. c Includes SHV-2a, SHV-5, SHV-12, and SHV-30. d Includes SHV-1, SHV-11, SHV-28, OKP-A/B, and LEN-1. e Includes TEM-3, TEM-11, TEM-24, TEM-52, and TEM-133. f Includes TEM-1 and TEM-2. g Total strains correctly identified, taking all the results into account. h Results obtained with classical PCR/sequencing. i Results obtained with the microarray. j % agreement between the two methods. k Sensitivity, 94%; specificity, 100%; positive predictive value, 100%; and negative predictive value, 98% for KPC detection. l Sensitivity, 100%; specificity, 100%; positive predictive value, 100%; and negative predictive value, 100% for CTX-M- detection. m Sensitivity, 100%; specificity, 100%; positive predictive value, 100%; and negative predictive value, 100% for SHV-ESBL detection. n Sensitivity, 93%; specificity, 100%; positive predictive value, 100%; and negative predictive value, 98% for TEM-ESBL detection. No. of strains % Downloaded from potential to detect an almost unlimited number of genes within one reaction mixture. Here, a new commercial DNA-based test, the Check-Points ESBL/KPC array, aimed at identifying TEM-, SHV-, and CTX-M-type ESBLs as well as KPC-type carbapenemases, was evaluated by comparing its performance with that of standard PCR on well-characterized reference strains and on 40 ESBL producers isolated at the Bicetre hospital from January to March MATERIALS AND METHODS Bacterial strains. Isolates were identified by conventional microbiological methods (API-20E; biomérieux, Marcy-l Etoile, France). Well-characterized CTX-M, TEM, SHV, and KPC producers (Table 1) were used to validate the novel detection assay. Several isolates that produced other resistance genes or that were wild type were used as negative-control strains. Forty ESBL-producing Enterobactericeae isolates were identified in clinical samples (one isolate per patient) during the first 3 months of 2009 at the Bicetre hospital (Kremlin- Bicêtre, France) (Table 2). Antimicrobial agents, susceptibility testing, and ESBL identification. Routine antibiograms were determined by the disk diffusion method on Mueller-Hinton (MH) agar (Bio-Rad, Marne-la-Coquette, France). The antimicrobial agents and their sources have been described elsewhere (21). MICs of ß-lactams were determined and interpreted as described previously (7). The double-disk synergy test, performed with ESC and ticarcillin-clavulanic acid disks on MH cloxacillin (250 g/ml)-containing agar plates (10), was used to evidence the presence of an ESBL in the clinical strains. Nucleic acid extractions. Whole-cell DNAs were extracted from reference strains or from clinical isolates using the QiaAmp DNA minikit (Qiagen, Les Ulis, France). PCR and Sanger DNA sequencing. PCR was performed on an ABI 2700 thermocycler (Applied Biosystems, Les Ulis, France) using laboratory-designed primers for detection and sequencing of the entire bla CTX-M -like, bla SHV, bla TEM, and bla KPC genes, as described previously (16, 22, 33). Two l of DNA extract (100 ng) was used as a template. PCR experiments were performed with 35 cycles consisting of 45 s of denaturation at 94 C, 45 s annealing at 55 C, and 60 s extension at 72 C. Both strands of the PCR products were sequenced with an ABI 3100 automated sequencer (Applied Biosystems). The nucleotide and deduced amino acid sequences were analyzed and compared to sequences available over the Internet at the National Center for Biotechnology Information website ( ncbi.nlm.nih.gov). Check-Points ESBL/KPC array. The Check-Points ESBL/KPC array uses a methodology called multiplex ligation detection reaction (LDR). Two probes are used: (i) a short one consisting of a target-specific sequence and a primer for PCR and (ii) a longer one consisting of a second target-specific sequence, a second primer for PCR, and, between, a ZIP code that is complementary to a unique oligonucleotide (czip) immobilized on the microarray. The two probes are ligated to each other when hybridized to a specific target sequence. A collection of DNA molecules are thus generated and are subsequently PCR amplified by means of the single pair of primers (34, 36). The PCR products are next detected by hybridization to a low-density DNA microarray. Positive hybridization is detected using a biotin label incorporated in one of the PCR primers (Fig. 1). ß-Lactamase gene identification relies on a series of probes targeting DNA markers whose sequence is specific to these enzymes and yield a unique microarray hybridization profiles to identify and discriminate between KPC, CTX-M, TEM, and SHV ESBL variants and TEM and SHV non-esbl variants. The five CTX-M groups could also be differentiated. The test allows single-tube processing, which simplifies the technical work associated with strain typing. The microarray setup is depicted in Fig. 2A. It includes a number of controls assessing the success of each critical step in the procedure, including ligation specificity and efficiency, PCR amplification, hybridization efficiency, label detection, and label quality. The detailed DNA sequences of the probes were not released by the manufacturer. Microarray results were read on a single-channel ATR03 reader consisting on October 30, 2018 by guest

3 3088 NAAS ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. DNA array results compared to classical PCR/sequencing obtained with the clinical isolates displaying a synergy image on disk diffusion antibiogram from the Bicetre Hospital from 2009 No. and species of isolates tested ß-Lactamase(s) identified a Microarray ESBL/KPC result 2 E. coli 1 Citrobacter koseri CTX-M-1 No KPC, CTXM-1 group 5 E. coli 1 Enterobacter cloacae CTX-M-15 No KPC, CTX-M-1 group b 1 E. coli CTX-M-32 No KPC, CTXM-1 group 1 Enterobacter cloacae CTX-M-9 No KPC, CTXM-9 group 2 E. coli CTX-M-14 No KPC, CTXM-9 group 5 E. coli CTX-M-1, TEM-1 No KPC, TEM non-esbl, CTX-M-1 group 1 E. coli CTX-M-14, TEM-1 No KPC, TEM non-esbl CTX-M-9 group 1 E. coli CTX-M-3, TEM-1 No KPC, TEM non-esbl, CTX-M1 group 1 E. coli CTX-M-27, TEM-1 No KPC, TEM non-esbl, CTX-M-9 group 5 E. coli 7 Enterobacter cloacae CTX-M-15, TEM-1 No KPC, TEM non-esbl, CTX-M-1 group 1 K. pneumoniae CTX-M-15, TEM-1, SHV-11 No KPC, TEM non-esbl, SHV non-esbl, CTX-M-1 group 1 K. pneumoniae CTX-M-15, SHV-28, SHV-1 No KPC, CTX-M-1 group, SHV non-esbl 2 E. coli TEM-52 No KPC, TEM ESBL 1 Enterobacter aerogenes TEM-24 No KPC, TEM ESBL 1 E. coli TEM-3 No KPC, TEM ESBL 1 E. coli TEM-1, SHV-12 No KPC, TEM non-esbl, SHV ESBL a ß-Lactamase genes were identified by PCR and sequencing. b E. cloacae isolate required a 1/10 dilution of DNA. basically of a CCD-based transmission detector (Clondiag, Jena, Germany). The reader was connected to a standard computer, and the data were processed by customized software supplied by the manufacturer (Check-Points, Wageningen, Netherlands). The software automatically translated the Check-Points ESBL/ KPC microarray data into presence or absence of a ß-lactamase gene. Reagents and procedures were supplied in a kit provided by the manufacturer (Check-Points). Briefly, 10 l of purified genomic DNA (5 to 50 ng/ l) was added to 8 l of a freshly prepared proprietary mix containing ligation probes and thermostable DNA ligase. The sample was heated in a MyCycler PCR instrument (Bio-Rad) for 3 min at 95 C, followed by 24 cycles of 0.5 min at 95 C and 5 min at 65 C, and then followed by a final denaturation at 98 C for 2 min. Next, 30 l of a second proprietary mix containing PCR primers, deoxynucleoside triphosphates, and thermostable polymerase were added. The sample was heated for 10 min at 95 C followed by 30 cycles of 0.5 min at 95 C, 0.5 min at 55 C, 0.5 min at 72 C, and a final denaturation step of 2 min at 98 C. The amplified ligation products (final volume, 48 l) were then subjected to DNA hybridization in a customized 1.5-ml ArrayTube (Clondiag) containing a 2- by 2-mm microarray spotted with DNA czip code oligonucleotides complementary to a set of unique sequences included in the LDR probes. Hybridization was performed using 10 l of each amplified reaction product supplemented with 300 l of preheated hybridization buffer and lasted for 30 min at 50 C under rotational shaking (400 rpm) in a thermo-mixer (Eppendorf, Hamburg, Germany). Thanks to the use of three independent probe sets, differing by ZIP code, each ArrayTube could detect three independent amplification reactions at once. Unbound DNA was washed away using two 5-min incubation steps with 300 l blocking buffer under the same conditions. Biotin label detection was then performed with 150 l of freshly prepared g/ml poly-horseradish peroxidase-conjugated streptavidin (Endogen, Rockford, IL) incubated for 15 min at 30 C and shaken at 400 rpm. Excess conjugate was washed away by two 5-min incubation steps using 300 l detection buffer under the same conditions. Finally, the detection reaction was initiated at room temperature by replacing the last washing solution with 150 l of ready-made TrueBlue peroxidase substrate (KPL, Gaithersburg, MD). DNA hybridization results were measured after 15 min on the reader. RESULTS The system was evaluated on a number of well-defined clinical isolates from different geographic origins and expressing several ß-lactamase genes. In addition, in the course of routine laboratory ESBL typing, the strains suspected to be ESBL positive (e.g., reduced susceptibility to ESC that is restored by clavulanic acid addition) were ESBL typed using standard PCR followed by sequencing and by the microarray system. The results are summarized in Tables 1 and 2. Detection of KPC. Using this microarray assay, all the KPC genes were detected in all the bacterial isolates tested, with the exception of one K. pneumoniae isolate (Table 1), for which failure to detect KPC was likely the result of plasmid instability, as evidenced by double zones around imipenem on a routine antibiogram. Furthermore, standard PCR on extracted DNA from that strain revealed a very weak KPC signal. Analysis of the plasmid carrying bla KPC-2 in that strain revealed a plasmid of 12 kb (21), with no partitioning functions, thus explaining its likely loss during bacterial culture without antibiotic pressure. For Pseudomonas aeruginosa, a 1:10 dilution of the DNA was often required in order to obtain a reproducible result. This is likely due to inhibitors present in the extracted DNA. However, for enterobacterial species, the quality of the DNA was sufficient, and the results were highly reproducible (Fig. 2B and C). Detection of ESBLs of the TEM, SHV, and CTX-M types in reference strains. Similarly, using this array approach, all the TEM (except one TEM-3)-, SHV-, and CTX-M-type ESBLs were reproducibly detected. Excellent detection was equally well obtained for KPC-positive strains harboring or not harboring ESBL genes. The TEM and SHV ESBLs could be distinguished from their non-esbl variants (Fig. 2D). The different CTX-M groups could be identified, and the different probes did not cross-react with the chromosomally encoded K1 ß-lactamases of Klebsiella oxytoca, which are genetically related to CTX-M (2). Furthermore, none of the other ß-lactamase genes cross-reacted with the probes used in the assay, suggesting an excellent specificity. Detection of the naturally encoded ß-lactamase in K. pneumoniae. When an SHV-ESBL is present, the software of array analysis indicates only the presence of the ESBL variant, but analysis by eye of the different arrays also revealed, for most of the K. pneumoniae isolates, the presence of spots specific to non-esbl variants. However, some Klebsiella isolates presented negative results even for non-esbl SHV variants in the absence of ESBL variants. These results could be explained by the heterogeneity of the natural ß-lactamase genes present in

4 VOL. 54, 2010 MICROARRAY FOR TEM, SHV, CTX-M ESBL, AND KPC DETECTION 3089 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. K. pneumoniae. Three families of the chromosomal ß-lactamase gene, bla SHV (found in 90% of K. pneumoniae clinical isolates), bla OKP, and bla LEN, sharing only 90% amino acid sequence identity, have been identified (12). bla OKP and bla LEN genes were not detected by the microarray. No plasmidencoded or ESBL variants of LEN or OKP have been reported yet, suggesting that these enzymes are not clinically relevant and that their detection failure is not a clinical issue. This was the case for five isolates that expressed either OKP-A/B or LEN. Identification of ESBLs in clinical isolates presenting a synergy image between ESC and ticarcillin-clavulanic acid disks. Over the first 3 months of 2009, 40 nonreplicate isolates were suspected to express an ESBL, based on synergy testing. Most of these isolates were identified as Escherichia coli isolates (Table 2). Using the microarray assay, the ESBLs present in these clinical isolates were investigated. The results obtained with the microarray were superimposable to those of standard PCR followed by sequencing of the entire genes. In one case, the signal for CTX-M-1 was weak and the software did not count the signal as positive. However, standard PCR confirmed the presence of CTX-M-1. Careful reading by eye of the array depicted the signal, and retesting of the sample with the microarray assay confirmed the presence of CTX-M-1. Overall, the Check-Points KPC/ESBL array was able to confirm the presence of an ESBL in these clinical isolates and to identify precisely the ESBL type in 7 h, while classical PCR followed by sequencing (with sometimes a need for transformation to separate ESBL and non-esbl variants of the SHV or TEM type) took between 3 and 10 days. DISCUSSION Identification of resistance to ESC and carbapenems in clinical samples is a major health issue and must be rapid for the management of patients, whatever mechanism of resistance is implicated. The most widespread and threatening resistance mechanisms are ESBLs and carbapenemases, especially KPClike enzymes. Detection of ESBLs based only on susceptibility testing is not easy, due to the variety of ß-lactamases and their variable expression. In routine laboratory situations, detection of ESC and carbapenem resistance is mainly based on phenotypic methods, but these methods often need technical changes and are time-consuming. Selective culture media (MacConkey and Drigalski agar supplemented with cefotaxime and/or ceftazidime) have been proposed for detection of GNB resistant to ESC (35). Media using chromogenic-medium-based substrates and selective antibiotics have been developed recently for the detection and presumptive identification of ESBL-producing Enterobacteriaceae directly from clinical specimens (32). Commercially available ESBL detection methods yield at most 90% accurate ESBL identification, since some ESBL producers may appear susceptible to several ESC (36). Enzymatic tests have also been proposed for identification of ESBL producers but cannot be implemented on a routine basis. Unambiguous identification of KPCs by phenotypic methods is relatively difficult (25). All together, these difficulties delay the response to an outbreak and/or its epidemiologic surveillance. Over the last 20 years, alternative strategies aimed at replacing or complementing traditional phenotypic methods have been proposed. Standard PCR followed by sequencing is still the most widely used approach, but its main pitfalls are the long delay and the necessity for TEM and SHV for conjugation or transformation experiments, since ESBL and non-esbl variants may exist in the same strain. Other molecular detection techniques for ESBLs of TEM and SHV types in general led to a few applications not really suited for a routine-based detection (1, 5, 19, 20, 24, 26, 31). Recently, real-time PCR coupled with pyrosequencing offered an interesting approach to detect CTX-M producers in pure cultures and for TEM and SHV enzymes (15, 22). This technique, even though rapid and highly discriminatory, has one main drawback, which is the

5 3090 NAAS ET AL. ANTIMICROB. AGENTS CHEMOTHER. Downloaded from 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 H (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 (TEM-1; panel II), and Enterobacter aerogenes BG (TEM-24; panel III). on October 30, 2018 by guest high cost of equipment (22). Multiplex PCR procedures coupled with inverse hybridization, with denaturing high-performance liquid chromatography (dhplc), or with TaqMan probe detection have been developed with pure cultures for detection of CTX-M enzymes (4, 11, 40). Similarly, real-time PCR procedures using TaqMan probes have been designed to detect KPC (13). The problems associated with these strategies include reproducibility, the requirement of specialized equip-

6 VOL. 54, 2010 MICROARRAY FOR TEM, SHV, CTX-M ESBL, AND KPC DETECTION 3091 ment, the high cost per sample analysis, the need for highly trained staff, and, for TEM and SHV, the necessity of further sequencing in order to differentiate non-esbl from ESBL variants. Finally, for TEM and SHV ESBLs, coexistence of both ESBL and non-esbl variants renders interpretation of sequencing results difficult, requiring additional experiments, such as transformation or conjugation. Overall, the Check-Points KPC/ESBL array correctly identified representatives of the four targeted gene classes in 119 of 125 reference isolates and was capable of distinguishing between ESBL and non-esbl variants of TEM and SHV, even when both variants were present in the same bacterial specimen. The Check-Points ESBL/KPC array was found to be robust, specific, and sensitive. Specificities and sensitivities of 100% were recorded for most gene classes. It gave satisfactory results on most enterobacterial extracts. However, for Pseudomonas spp. and nonfermenters, a 1:10 dilution was often necessary even on purified DNA. The analysis time (7 to 8 h) was relatively short compared to the barely predictable time scale of classical PCR followed by sequencing. The single-tube processing of the samples was found particularly convenient compared to other multiplexed analysis systems. The reading of the microarray results required a very simple device hosted in a standard molecular biology laboratory and required no particular technical expertise. The system could differentiate TEM- ESBL and SHV-ESBL from non-esbl variants. Taken together, the three major ESBLs and the currently most important class A carbapenemase, KPC, have been successfully identified. This assay may become a key technique in epidemiology of resistance genes and in infection control follow-up, where large collections of isolates need to be characterized. It may also be used as a first-line experiment to rapidly identify the resistance determinants present in clinical samples in order to amplify and sequence only the ESBL genes. The open design of the ligation-mediated detection method on the one hand and of the ZIP-code microarray on the other renders the system easily improvable to reach the sensitivity level required for the typing of hundreds of different resistance genes. Future releases of this microarray should therefore include additional genetic markers to increase the number of detected genes, such as those of minor ESBLs, plasmid-encoded cephalosporinases, OXA-48, and metallo-ß-lactamases. The current system sensitivity is sufficient for the preliminary identification of ESBLs and KPC in GNB. Conclusion. Laboratory detection of ESBL and KPC producers remains a challenge for the microbiology laboratory and is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. An efficient assay should be fast, reliable, able to detect the most important ESBLs, and able to distinguish between ESBL and non-esbl TEM and SHV variants. The Check-Points ESBL/KPC array may fulfill these criteria. ACKNOWLEDGMENTS This work was funded by a grant from the Ministère de l Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, Paris, by the Assistance Publique-Hôpitaux de Paris, France, by the European Community (7th PCRD, TROCAR contract HEALTH-F ), and by INSERM, France. We also thank Check-Points for providing the material necessary for this study. REFERENCES 1. Arlet, G., G. Brami, D. Decre, A. Flippo, O. Gaillot, P. H. Lagrange, and A. Phillipon Molecular characterization by PCR-restriction fragment length polymorphism of TEM ß-lactamases. FEMS Microbiol. Lett. 134: Bauernfeind, A., I. Stemplinger, R. Jungwirth, S. Ernst, and J. M. 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