ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2011, p. 4457–4460 0066-4804/11/$12.00 doi:10.1128/AAC.00353-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 9
Multicenter Evaluation of a New DNA Microarray for Rapid Detection of Clinically Relevant bla Genes from -Lactam-Resistant Gram-Negative Bacteria䌤 Pierre Bogaerts,1* Andrea M. Hujer,2,3 Thierry Naas,4 Roberta Rezende de Castro,1 Andrea Endimiani,2,3 Patrice Nordmann,4 Youri Glupczynski,1 and Robert A. Bonomo2,3,5,6 Laboratoire de Bacte´riologie, Cliniques Universitaires UCL de Mont-Godinne, B-5530 Yvoir, Belgium1; Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio2; Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio3; Service de Bacte´riologie-Virologie, INSERM U914: Emerging Resistance to Antibiotics, Ho ˆ pital de Biceˆtre, Assistance Publique-Ho ˆ pitaux de Paris, Faculte´ de Me´decine Paris-Sud, 94275 Le Kremlin-Biceˆtre, France4; and Pharmacology5 and Molecular Biology and Microbiology,6 Case Western Reserve University School of Medicine, Cleveland, Ohio Received 16 March 2011/Returned for modification 22 May 2011/Accepted 26 June 2011
A new commercial low-density microarray which identifies common extended-spectrum -lactamase plasmid-mediated cephalosporinase genes, as well as carbapenemase (blaKPC and blaNDM) genes, was evaluated. We tested 207 clinical and reference/collection isolates of the Enterobacteriaceae possessing different bla genes. Overall, the sensitivity and specificity of the microarray were 100% for the detection of the plasmid-mediated blaAmpC, blaKPC, and blaNDM genes using bla gene sequencing as the reference method. Resistance to -lactams, including carbapenems, is rapidly spreading and is related to the worldwide dissemination of genes encoding extended-spectrum -lactamases (ESBLs), carbapenemases, and plasmid-mediated AmpC -lactamases (pAmpCs) (3, 8). Class C cephalosporinases (AmpCs), which belong to group 1 -lactamases (4), are encoded primarily in the chromosome of many species of the Enterobacteriaceae and confer resistance to penicillins, cephalosporins (narrow and extended spectrum), cephamycins (cefoxitin and cefotetan), and monobactams (aztreonam). Moreover, when associated with outer membrane protein (OMP) or porin deficiencies, AmpC -lactamase overproduction can also confer resistance to carbapenems (9, 13). Plasmid-mediated blaAmpC -lactamases (pAmpC) are now increasingly reported worldwide (7, 12, 15, 16) and are categorized into six groups: (i) the CMY-2 group, (ii) the MIR-1 and ACT-1 group, (iii) the DHA group, (iv) a fourth group represented by ACC-1, and two clusters represented by (v) CMY-1 (also called MOX-1) and (vi) FOX-1. These various pAmpCs likely escaped from the chromosome of natural AmpC-producing species of the Enterobacteriaceae (7). Only molecular techniques are able to confirm the presence of pAmpC -lactamases, which is of particular importance for epidemiological purposes and for the reporting of cephalosporin susceptibility testing (17). In the present study, we tested the Check-Points ESBL/ AmpC/KPC/NDM-1 assay (Check-MDR CT101; Check-Points B.V., Wageningen, The Netherlands). In comparison to the
former ESBL-KPC array (Check-KPC ESBL) recently evaluated (5, 6, 10), the present array targets 6 additional groups of pAmpC genes and the blaNDM-1 metallo--lactamase gene (Fig. 1). The system was evaluated in parallel at three centers, in Yvoir (Belgium), in Cleveland (Ohio), and in Paris (France). A total of 207 isolates of the Enterobacteriaceae (200 clinical isolates from 3 different geographic origins, 1 nontransformed Escherichia coli isolate, DH10B, 5 E. coli DH10B isolates expressing cloned blaAmpC genes, and 1 Klebsiella pneumoniae isolate, ATCC 700603, expressing SHV-18) possessing different bla genes were included. Species identification was carried out using matrix-assisted laser desorption ionization–time-offlight (MALDI-TOF) mass spectrometry on a Microflex LT (Bruker Daltonics) or Microscan (Siemens) instrument or by using a Vitek2 system (bioMe´rieux). These strains had been well characterized or were characterized for this study with respect to their bla genes by PCR amplification and standard DNA sequencing as previously described (1, 2, 6, 10, 14) (Tables 1 and 2) or by PCR sequencing using an external sequencing service (Macrogen Inc., Seoul, South Korea). This collection included 24 chromosomal blaAmpC-producing isolates of the Enterobacteriaceae (Table 2) known to carry the progenitors of pAmpCs. These blaAmpC-producing members of the Enterobacteriaceae were screened in order to evaluate the reactivities of the probes targeting the pAmpC genes versus their chromosomal progenitors. The whole microarray detection process requires 7 h of effort and was performed according to the manufacturer’s instructions. In brief, whole-cell DNA was purified from 0.5 McFarland bacterial suspension using the EasyMag system (bioMe´rieux) or the DNeasy blood and tissue kit (Qiagen) (45 min). Twenty to forty nanograms of the DNA extract was used for a first ligation step (150 min), followed by a PCR amplification step (90 min). PCR products were then hybridized to
* Corresponding author. Mailing address: Laboratoire de Bacte´riologie, Cliniques Universitaires UCL de Mont-Godinne, Av Dr Therasse 1, B-5530 Yvoir, Belgium. Phone: 32-81-42-32-41. Fax: 32-81-4232-04. E-mail:
[email protected]. 䌤 Published ahead of print on 11 July 2011. 4457
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FIG. 1. Typical DNA microarray pictures obtained with the “Check-MDR CT101” 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 for one strain and consists of control spots and specific marker spots, which are numbered from 1 to 96. The results are automatically interpreted by the software provided by the manufacturer. (A) Theoretical display of the array probes for strain 1 (I), strain 2 (II), and strain 3 (III); black dots refer to targets detected in panel B. (B) Array results for E. coli CP157 (TEM-1, FOX-3; panel I), E. coli CP27 (NDM-1, TEM-1, CTX-M-15, and CMY-58; panel II), and K. pneumoniae CP07 (SHV-11, DHA-1; panel III).
the array and colorimetrically revealed (90 min). The results were read in a single-channel ATR03 array tube reader (Alere, Cologne, Germany) and automatically interpreted by the software provided by the manufacturer (15 min). Using the Check-MDR CT101 assay, all the blaAmpC genes were correctly detected in all 87 pAmpC-producing isolates tested (Table 1). When testing the array against 24 chromosomally encoded AmpC-producing isolates of the Enterobacteriaceae and one E. coli isolate in which chromosomal AmpC of Enterobacter cloacae P99 (E. coli P99) was cloned (Table 2), we observed that the microarray did frequently detect the blaAmpC progenitor. In particular, the blaCMY-2 gene was detected in 4 out of 10 Citrobacter freundii isolates, blaACT/MIR was identified in 5 out of 7 E. cloacae/Enterobacter asburiae isolates and in E. coli P99, blaDHA was detected in all four Morganella morganii isolates and blaACC in 1 out of 2 Hafnia alvei isolates, but blaCMY-1 was not detected in Aeromonas hydrophila. Hence, we recommend identifying clinical isolates to the species level before testing them on the array in order to ensure the accuracy of plasmid-carried blaAmpC detection. Provided that the test was not performed on chromosomally encoded AmpC-producing species, specificities, sensitivities, and positive and negative predictive values of 100% were recorded for representative isolates of the six plasmid-carried blaAmpC gene families tested. Regarding carbapenemases, KPC (K. pneumoniae [n ⫽ 12], E. coli [n ⫽ 3], and C. freundii [n ⫽ 1]; not shown) and NDM-1 (K. pneumoniae [n ⫽ 13], E. coli [n ⫽ 2], including 10 concomitantly harboring pAmpC genes; Table 1) were correctly detected in all 31 strains tested (100% sensitivity and specificity), underscoring the ability of the Check-MDR CT101 array to
detect some of the most worrisome carbapenemase genes recovered in the Enterobacteriaceae. We did not observe falsepositive results. Regarding detection of blaESBL genes, contrary to the case with the former ESBL array, when blaTEM or blaSHV nonESBLs are simultaneously present with a second blaTEM or blaSHV ESBL variant, respectively, the new array reports only the presence of the blaESBL, ignoring the wild-type gene. Our results (not shown) confirmed those previously published (6, 10), with 100% specificity for the detection of blaTEM, blaSHV, and bla-CTX-M genes (94 blaTEM non-ESBL genes and 17 blaTEM-ESBL, 52 blaSHV non-ESBL or blaLEN, 24 blaSHV-ESBL, and 41 blaCTX-M genes) and a sensitivity varying between 92% for blaSHV non-ESBL or blaLEN (principally due to the lack of detection of blaLEN), 96% for blaSHV-ESBL (blaSHV-38 is not detected by the array [10]), and 100% for the blaTEM-ESBL, blaTEM non-ESBL, and blaCTX-M genes. Owing to its rapid and precise analytical performance, this platform appears well suited for use in epidemiological or infection control studies in which large collections of isolates require characterization. The fact that multiple resistance genes were correctly identified whatever the complex genetic background of the tested isolates (presence of multiple ESBL genes and of the blaNDM-1 or blaKPC gene in addition to the various blaAmpC genes) confirms that the Check-Points array technology is a highly accurate tool for detection of coexistent bla genes. Use of this technology on a routine basis is still hampered by the low prevalence of pAmpC- and carbapenemase-producing isolates in most countries and by the length of the detection process (7 h). However, the system was shown to be flexible and clinically responsive since new important targets
4459 MICROARRAY FOR DETECTION OF AmpC -LACTAMASES AND NDM-1 VOL. 55, 2011
TABLE 1. DNA array results for various clinical isolates harboring pAmpC- and NDM-1-encoding genes
Array
1 0 0 0 0 0 0
PCR
1
1 0 0 0 0 0 0
Array
3
0 3 0 0 0 0 0
PCR
3
0 3 0 0 0 0 0
Array
3
2 1 0 0 0 0 0
PCR
3
2 1 0 0 0 0 0
Array
15
2 13 0 0 0 0 0
PCR
15
2 13 0 0 0 0 0
No. of isolates with detection of gene groupj
PCR
2 3 0 0 2 0 0
1
Array
blaNDM-1k
Array
2 3 0 0 2 0 0
7
blaCMY-1 like/MOXf
PCR
1 8 0 0 0 0 0
7
blaACT/MIRe
Arrayi
1 8 0 0 0 0 0 9
blaACC
PCRh
50 12 1 0 0 1 0 9
blaFOXd
No. of isolatesa
50 12 1 0 0 1 0 64
blaDHAc
Species
89 66 8 7 5 1 6 64
blaCMY-2 likeb
Escherichia coli Klebsiella pneumoniae Proteus mirabilis Enterobacter aerogenes Klebsiella oxytoca Salmonella spp. Othersg 182
Total
a Includes control isolates but does not include 25 strains harboring pAmpC progenitors. For E. coli, includes 84 clinical isolates and 4 E. coli isolates expressing cloned CMY-2-like-encoding genes and 1 E. coli recipient strain. b Includes CMY-2 (n ⫽ 53), CMY-30 (n ⫽ 2), CMY-32 (n ⫽ 2), CMY-33 (n ⫽ 1), CMY-42 (n ⫽ 1), CMY-44 (n ⫽ 1), CMY-58 (n ⫽ 1), CMY-60 (n ⫽ 1), CMY-61 (n ⫽ 1), and CMY-62 (n ⫽ 1). c Includes DHA-1 (n ⫽ 6), DHA-2 (n ⫽ 2), and DHA-7 (n ⫽ 1). d Includes FOX-3 (n ⫽ 1) and FOX-5 (n ⫽ 6). e Includes MIR-1 (n ⫽ 1) and ACT-1 (n ⫽ 2). f Includes CMY-1 (n ⫽ 1) and CMY-10 (n ⫽ 2). g Includes Citrobacter koseri (n ⫽ 2), Serratia marcescens (n ⫽ 2), Citrobacter braakii (n ⫽ 1), and Providencia stuartii (n ⫽ 1). h Results obtained with PCR/sequencing. i Results obtained with the Check-MDR CT101 microarray. One hundred percent agreement between the two methods for all gene groups. For all pAmpC- and NDM-1-producing isolates: sensitivity, 100%; specificity, 100%; positive and negative predictive value, 100%. Fifteen isolates concomitantly harboring CTX-M-15, 14 harboring TEM-1, 11 harboring non-ESBL SHV, 1 harboring SHV-12, and 10 harboring pAmpC (CMY-2-like) genes. j
k
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TABLE 2. DNA array results for 25 isolates harboring chromosomal AmpC-encoding genes Species carrying plasmid-encoded AmpC progenitor (AmpC type)
No. of isolates tested
blaCMY-2
blaDHA
blaFOX
blaACC
blaACT/MIR
blaCMY-1
Citrobacter freundii (CMY-2) Enterobacter cloacae (MIR) ⫹ E. coli P99 Enterobacter asburiae (ACT) Morganella morganii (DHA) Aeromonas hydrophila (CMY-1) Hafnia alvei (ACC)
10 6 1 4 2 2
4 0 0 0 0 0
0 0 0 4 0 0
0 0 0 0 0 0
0 0 0 0 0 1
0 4 1 0 0 0
0 0 0 0 0 0
No. of isolates with array detection of gene group
were rapidly incorporated into the assay by the manufacturer. We anticipate that additional targets, such as oxacillinase-coding genes and, especially for nonfermenters, minor ESBLs (BEL, VEB, PER, and GES) (11) and OXA-carbapenemases could readily be added and validated whenever the need arises. This work was supported in part by grants from the Belgian Antibiotic Policy Coordination Committee (BAPCOC), Ministry of Public Health, Belgium, by a grant from the European Community (TEMPOtest-QC, HEALTH 2009-241742), by INSERM, France, by a grant from the Ministe`re de l’Education Nationale et de la Recherche (UPRES-EA3539), Universite´ Paris XI, Paris, France, by the National Institutes of Health (grants AI072219 and AI063517 to R.A.B.), and by the Geriatric Research Education and Clinical Center VISN 10 (to R.A.B.). We thank Check-Points for providing the material necessary for the study and Aneta Karczmarek for technical support. REFERENCES 1. Bogaerts, P., et al. 2010. Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing KPC-2 carbapenemase in Belgium. J. Antimicrob. Chemother. 65:361–362. 2. Bogaerts, P., et al. 2009. Emergence of extended-spectrum-AmpC-expressing Escherichia coli isolates in Belgian hospitals. J. Antimicrob. Chemother. 63:1073–1075. 3. Bush, K. 2010. Bench-to-bedside review: the role of beta-lactamases in antibiotic-resistant Gram-negative infections. Crit. Care 14:224. 4. Bush, K., and G. A. Jacoby. 2010. Updated functional classification of betalactamases. Antimicrob. Agents Chemother. 54:969–976. 5. Cohen, S. J., et al. 2010. Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. J. Antimicrob. Chemother. 65:1377–1381.
% of progenitors detected by array
40 67 100 100 0 50
6. Endimiani, A., et al. 2010. Evaluation of a commercial microarray system for detection of SHV-, TEM-, CTX-M-, and KPC-type beta-lactamase genes in Gram-negative isolates. J. Clin. Microbiol. 48:2618–2622. 7. Jacoby, G. A. 2009. AmpC beta-lactamases. Clin. Microbiol. Rev. 22:161– 182. 8. Livermore, D. M. 2009. Has the era of untreatable infections arrived? J. Antimicrob. Chemother. 64(Suppl. 1):i29–i36. 9. Mammeri, H., H. Guillon, F. Eb, and P. Nordmann. 2010. Phenotypic and biochemical comparison of the carbapenem-hydrolyzing activities of five plasmid-borne AmpC beta-lactamases. Antimicrob. Agents Chemother. 54: 4556–4560. 10. Naas, T., G. Cuzon, H. Truong, S. Bernabeu, and P. Nordmann. 2010. 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. Antimicrob. Agents Chemother. 54:3086–3092. 11. Naas, T., L. Poirel, and P. Nordmann. 2008. Minor extended-spectrum beta-lactamases. Clin. Microbiol. Infect. 14(Suppl. 1):42–52. 12. Pitout, J. D., D. B. Gregson, D. L. Church, and K. B. Laupland. 2007. Population-based laboratory surveillance for AmpC beta-lactamase-producing Escherichia coli, Calgary. Emerg. Infect. Dis. 13:443–448. 13. Poirel, L., J. D. Pitout, and P. Nordmann. 2007. Carbapenemases: molecular diversity and clinical consequences. Future Microbiol. 2:501–512. 14. Rodriguez-Villalobos, H., et al. 2011. Trends in production of extended-spectrum beta-lactamases among Enterobacteriaceae of clinical interest: results of a nationwide survey in Belgian hospitals. J. Antimicrob. Chemother. 66:37–47. 15. Sidjabat, H. E., et al. 2009. Clinical features and molecular epidemiology of CMY-type beta-lactamase-producing Escherichia coli. Clin. Infect. Dis. 48: 739–744. 16. Tan, T. Y., L. S. Ng, J. He, and L. Y. Hsu. 2010. CTX-M and ampC beta-lactamases contributing to increased prevalence of ceftriaxone-resistant Escherichia coli in Changi General Hospital, Singapore. Diagn. Microbiol. Infect. Dis. 66:210–213. 17. Tenover, F. C., et al. 2009. Identification of plasmid-mediated AmpC betalactamases in Escherichia coli, Klebsiella spp., and proteus species can potentially improve reporting of cephalosporin susceptibility testing results. J. Clin. Microbiol. 47:294–299.
