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1 ORIGINAL ARTICLE /j x Development and clinical validation of a molecular diagnostic assay to detect CTX-M-type b-lactamases in Enterobacteriaceae J. D. D. Pitout 1,2,3, N. Hamilton 1, D. L. Church 1,2,4, P. Nordmann 5 and L. Poirel 5 1 Division of Microbiology, Calgary Laboratory Services, 2 Department of Pathology and Laboratory Medicine, 3 Department of Microbiology and Infectious Diseases, 4 Department of Medicine, University of Calgary, Calgary, Alberta, Canada and 5 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique Hôpitaux de Paris, Faculté de Médecine Paris-Sud, University Paris XI, Le Kremlin-Bicêtre, France ABSTRACT Enterobacterial isolates producing CTX-M b-lactamases have recently emerged worldwide in the community and hospital settings. Because of the significant public health implications, the spread of organisms producing CTX-M enzymes merits close monitoring with enhanced surveillance efforts. A molecular diagnostic assay using two different sets of primers simultaneously for the detection of all bla CTX-M -like b-lactamase genes was developed. This assay repeatedly demonstrated 100% sensitivity, specificity and positive and negative predictive values for detecting different CTX-M enzymes in well-characterised strains that included producers of VEB-, TEM- and SHV-type extended-spectrum b-lactamases (ESBLs) and plasmid-mediated AmpC enzymes. The majority ( ; 55%) of ESBL-producing enterobacterial isolates recovered in the Calgary Health Region during 2003 and 2004 were positive for bla CTX-M genes, including 81 (61%) positive for the CTX-M-9 group, 49 (37%) for the CTX-M-1 group, and two (2%) for the CTX-M-2 group. The CTX-M-specific PCR assay was reproducible and easy to use. It can be introduced in a clinical or reference laboratory to track and monitor the spread of organisms producing CTX-M enzymes in the community and hospital settings. Keywords b-lactamase, CTX-M enzymes, detection, Enterobacteriaceae, PCR, surveillance Original Submission: 20 June 2006; Revised Submission: 21 August 2006; Accepted: 13 September 2006 Clin Microbiol Infect 2007; 13: INTRODUCTION Enterobacteriaceae, especially Klebsiella spp. producing extended-spectrum b-lactamases (ESBLs) belonging to the SHV and TEM groups, have been recognised since the 1980s as a major cause of hospital-acquired infections [1]. More recently, Enterobacteriaceae (mostly Escherichia coli) producing novel ESBLs, e.g., the CTX-M enzymes, have emerged within the hospital and community settings as an important cause of urinary tract infection [2,3]. The CTX-M b-lactamases, of which there are now more than 40 types, can be divided into five groups based on their amino-acid identities [4]: the CTX-M-1 group (including CTX-M-1, Corresponding author and reprint requests: J. D. D. Pitout, Division of Microbiology, Calgary Laboratory Services, #9, 3535 Research Road NW, Calgary, Alberta, Canada T2L 2K8 johann.pitout@cls.ab.ca -3, -10, -12, -15, -28 and -30, and FEC-1); the CTX-M-2 group (including CTX-M-2, -4, -5, -6, -7 and -20, and Toho-1); the CTX-M-8 group (including CTX-M-8); the CTX-M-9 group (including CTX-M-9, -13, -14, -16, -17, -19, -21, -24 and -27, and Toho-2); and the CTX-M-25 group (including CTX-M-25 and -26). Surveys from several countries, e.g., Canada [5,6], Spain [7 9], France [10], Korea [11] and the UK [12], have shown that CTX-M-producing Esch. coli strains, isolated from hospital and community settings, often exhibit co-resistance to trimethoprim sulphamethoxazole, tetracycline, gentamicin and ciprofloxacin. Woodford et al. [12] identified an epidemic clone of Esch. coli producing CTX-M-15 that had become widely distributed throughout the UK [12], while a Canadian study described a CTX-M-14-producing strain of Esch. coli that was the cause of a community-wide clonal outbreak of urinary tract Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 292 Clinical Microbiology and Infection, Volume 13 Number 3, March 2007 infection during 2000 and 2001 [13]. Similar findings involving Esch. coli strains producing CTX-M-9 and CTX-M-14 have been reported from Spain [14,15]. The significant public health implications, including the treatment of communityacquired urinary tract infection, mean that the spread of organisms producing ESBLs (particularly CTX-M enzymes) merits close monitoring with enhanced surveillance, and efforts should be made to prevent the spread of organisms producing these enzymes within the hospital and community settings. Several studies have described molecular approaches that allow screening of ESBL-positive organisms for the presence of different CTX-M genes. Amplification and sequencing of bla CTX-M genes is time-consuming and expensive, and involves multiple reactions with different primers for each specific gene [16,17]. Some studies have utilised amplification of a universal DNA fragment specific for most of the different groups of CTX-M b-lactamases [18,19], and a multiplex PCR that differentiates between the five CTX-M phylogenetic groups has also been described [20]. Unfortunately, none of these methods has been verified extensively or validated in routine use. Therefore, the present study was designed to develop, validate and verify a molecular detection assay for the simultaneous detection of strains carrying different types of bla CTX-M b-lactamase genes, and to investigate the extent to which genes encoding these enzymes were present among ESBL-producing isolates of Enterobacteriaceae from clinical specimens in the Calgary Health Region, Canada, during 2003 and MATERIALS AND METHODS Bacteria Consecutive non-duplicate isolates of all Enterobacteriaceae (one per patient; n = 240) collected at Calgary Laboratory Services during January 2003 and December 2004 were used in this study. These organisms were screened for ESBL production and then investigated for the presence of CTX-M b-lactamases. Isolates were identified to the species level with the Vitek AMS (biomérieux Vitek Systems Inc., Hazelwood, MO, USA). Strains (n = 28) with well-characterised b-lactamases were used as positive and negative controls for the verification and validation of the PCR assay (Table 1). An additional group (n = 14) of AmpC-producing Esch. coli strains, isolated recently in the Calgary Health Region, were also included; these isolates were resistant to cefoxitin, cefotaxime and ceftazidime, and tested positive for AmpC cephalosporinases using the combination of the AmpC Strain Organism b-lactamase Group a Primers CTXMA1 A2 b CTX825F R b Reference or source Table 1. Control strains producing well-characterised b-lactamases CF2 Enterobacter cloacae CTX-M [31] Rio-4 Proteus mirabilis CTX-M [32] VER-1 Ent. cloacae CTX-M [31] Cfr Citrobacter freundii CTX-M [33] 34 Salmonella typhimurium CTX-M [34] Rio-3 Enterobacter aerogenes CTX-M [32] 785D Escherichia coli CTX-M [35] EC Esch. coli CTX-M [36] EC Esch. coli CTX-M [37] CF1 Esch. coli CTX-M [31] Eco Esch. coli CTX-M [38] N Esch. coli CTX-M [39] Rio-6 Esch. coli CTX-M [40] BM4493 Klebsiella pneumoniae CTX-M [41] ILT-2 K. pneumoniae CTX-M [42] ILT-3 K. pneumoniae CTX-M [42] ESBL530 Esch. coli CTX-M [43] Cf29 C. freundii CTX-M [44] CT1 Esch. coli Toho [45] 1A Esch. coli VEB-1 [46] S1 Esch. coli SHV-2 [47] S2 Esch. coli SHV-7 [47] T1 Esch. coli TEM-3 [47] T4 Esch. coli TEM-10 [47] T5 Esch. coli TEM-50 [47] A1 Esch. coli MOX-1 [47] A2 Esch. coli FOX-1 [47] A3 Salmonella spp. CMY-2 [48] C1 Ent. cloacae NMC-A [47] C2 K. pneumoniae KPC-1 [47] a Group 1 includes CTX-M-1, -3, -10, -11, -12, -15, -22, -23, -28, -29 and -30; group 2 includes CTX-M-2, -4, -5, -6, -7 and -20, and Toho-1; group 8 includes CTX-M-8; group 9 includes CTX-M-9, -13, -14, -16, -17, -18, -19, -21 and -27, and Toho-2; and group 25 includes CTX-M-25 and -26. b PCR primers for CTX-M groups (see Materials and methods).

3 Pitout et al. Molecular diagnostic assay for CTX-M enzymes 293 b-lactamase inhibitor Syn2190 and cefotetan disks, as described previously [21]. Antimicrobial susceptibility testing MICs of piperacillin tazobactam, imipenem, gentamicin, tobramycin, trimethoprim sulphamethoxazole and ciprofloxacin were determined using the Vitek AMS. Throughout this study, results were interpreted using CLSI (NCCLS) criteria for broth dilution [22]. The quality control strains used for this part of the study were Esch. coli ATCC and Esch. coli ATCC Screening for and confirmation of ESBLS The presence of ESBLs in clinical isolates of Esch. coli and Klebsiella spp. was investigated using the CLSI (NCCLS) criteria for ESBL screening and disk confirmation tests [22]. Clinical isolates of Enterobacteriaceae other than Esch. coli and Klebsiella spp. were investigated for ESBL production using the modified double-disk test [23]. Disks for ESBL confirmation tests were obtained from Oxoid (Nepean, Ontario, Canada). Klebsiella pneumoniae ATCC and Esch. coli ATCC were used as positive and negative controls, respectively. b-lactamase gene identification Template DNA was prepared from an overnight culture (18 24 h) on a Mueller Hinton plate. Two colonies were resuspended in 100 ll of distilled water and the cells were lysed by heating at 95 C for 10 min. Cellular debris was removed by centrifugation at g for 2 min, and the supernatant was used as a source of template DNA for amplification. PCR for the simultaneous detection of bla CTXM b-lactamase genes was performed on a 9700 thermal cycler (Applied Biosystems, Norwalk, CT, USA) with Platinium Taq DNA polymerase kits and a 2 mm dntp mix (Invitrogen, Carlsbad, CA, USA). Reaction mixtures comprised 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 1.5 mm MgCl 2, 0.25 mm datp, dgtp and dctp (Invitrogen), 0.75 mm dutp (Roche Diagnostics, Laval, Quebec, Canada), 0.1 lm CTX-MA1, CTX-MA2, CTX825-F and CTX-825R primers (see below), lm rrs-1 primer and rrs-2 primer [24], U of uracil-n-glycosylase and 2.5 U of Platinum Taq, in a total volume of 22 ll. Following the addition of 3 ll of sample lysate to the reaction mixture, the PCR program comprised 10 min at 37 C and 6 min at 94 C, followed by 30 cycles of 92 C for 1 min, 55 C for 1 min and 72 C for 1 min, followed by 72 C for 7 min. The PCR products were then stored at 4 C until analysis (0.1 volume) by electrophoresis in agarose 1.5% w v gels in 0.5 TBE buffer (1 TBE is 89 mm Tris, 89 mm boric acid, 2 mm EDTA). The gels were stained with ethidium bromide and the PCR products were visualised with UV light. amplification of CTX-M types from groups CTX-M-8 and CTX- M-25 were CTX825F (5 -CGCTTTGCCATGTGCAGCACC) and CTX825R (5 -GCTCAGTACGATCGAGCC), yielding a 307-bp amplicon [26]. Verification of the assay The accuracy (i.e., clinical sensitivity and specificity) of the test was investigated using strains with well-characterised resistance mechanisms (Table 1) as described previously [26]. Validation of the assay The performance of the assay was evaluated over a period of 3 months [27], including the addition of a 320-bp internal control (16S rrna, rrs) to the reaction mixture [24], and the repeated testing of well-characterised strains producing known CTX-M b-lactamases, using different personnel and amplification instruments, including a 9800 Thermal Cycler (Applied Biosystems). Results for the clinical isolates were validated by testing all isolates that gave negative results with the duplex CTX-M PCR by analytical isoelectric focusing (aief) and PCRs for bla TEM and bla SHV [28], while positive isolates were further tested by aief and CTX-M group-specific primers [26]. RESULTS Verification of the assay for bla CTX-M genes The primers used in this study were verified using DNA templates prepared from control strains with specific CTX-M b-lactamases or strains producing b-lactamases other than CTX- M enzymes (Table 1). The CTX-MA1 A2 primers showed 100% sensitivity, specificity and positive and negative predictive values for detecting CTX- M-1, -2, -3, -5, -9, -10, -14, -15, -16, -17, -19 and -30, while the CTX825F R primers showed 100% sensitivity, specificity and positive and negative predictive values for detecting CTX-M-8 and -25. Both sets of primers were used simultaneously and did not cross-react with strains producing VEB-1, TEM-3, -10 and -52, SHV-2, -7 and -12, KPC-1, NMC-A, MOX-1, CMY-2 and FOX-1 (Table 1). The same results were obtained with different species of Enterobacteriaceae (Tables 1 and 2). Primers The primers used for PCR amplification of CTX-M types from groups CTX-M-1, CTX-M-2 and CTX-M-9 were CTX-MA1 (5 - SCSATGTGCAGYACCAGTAA) and CTX-MA2 (5 -CCGCRA- TATGRTTGGTGGTG), yielding a 450-bp amplicon [25] (S = G or C; Y = C or T; R = A or G). These are degenerate primers that are a mixture of different primers in equal amounts to ensure optimal binding capacity. The primers used for PCR Validation of the assay for bla CTX-M genes The assay procedure was validated by adding the rrs internal control and by repeating the procedure five times on consecutive days with the control strains shown in Fig. 1. To ensure that the assay would be suitable for routine diagnostic

4 294 Clinical Microbiology and Infection, Volume 13 Number 3, March 2007 Species a n CTX-M-1 CTX-M-2 CTX-M-9 CTX-M-8 Escherichia coli (38%) 2 (2%) 75 (60%) 0 0 Klebsiella spp. 7 1 (14%) 0 6 (86%) 0 0 Enterobacter cloacae 1 1 (100%) CTX-M-25 Table 2. Group-specific CTX-M b-lactamases detected using groupspecific primers [26] among recent clinical isolates of CTX-M-positive Enterobacteriaceae a Positive with the CTX-M PCR assay. b Group 1 includes CTX-M-1, -3, -10, -11, -12, -15, -22, -23, -28, -29 and -30; group 2 includes CTX-M-2, -4, -5, -6, -7 and -20, and Toho-1; group 8 includes CTX-M-8; group 9 includes CTX-M-9, -13, -14, -16, -17, -18, -19, -21 and -27, and Toho-2; and group 25 includes CTX-M-25 and -26. L H N1 N2 N3 P1 P2 P3 P4 P5 N4 N5 N6 N7 L 500 bp rrs 300 bp Fig. 1. PCR assay for the detection of bla CTX-M genes. Lanes: L, 100-bp ladder; H, distilled water; N1, T1 (TEM-1); N2, T4 (TEM-10); N3, A1 (MOX-1); P1, CF2 (CTX-M-1); P2, Rio-4 (CTX-M-2); P3, Rio-3 (CTX-M- 8); P4, CF1 (CTX-M-14); P5, ESBL530 (CTX-M-25); N4, S1 (SHV-2); N5, T1 (TEM-3); N6, A1 (MOX-1); and N7, A2 (KPC-1). rrs indicates the 16S rrna internal control. Specific marker sizes in base pairs are shown on the left. More details of the individual strains are given in Table 1. use, the procedure was repeated an additional five times using different thermocyclers on different days with different personnel. The assay performed accurately on each occasion. Clinical bacterial isolates Of 240 ESBL-producing Enterobacteriaceae isolated during the study period, 217 (90%) were Esch. coli, 12(5%) were Klebsiella spp., six (3%) were Salmonella spp., two (1%) each were Citrobacter freundii and Enterobacter cloacae, respectively, and one was Citrobacter koseri. The majority of isolates (211; 88%) were recovered from urine, 14 (6%) from blood, six (3%) from stool, and the remaining nine (4%) from miscellaneous other specimens. Of the 240 clinical isolates included in this study, 132 (55%) were resistant to trimethoprim sulphamethoxazole, 20 (8%) to piperacillin tazobactam, 95 (40%) to tobramycin, 98 (41%) to gentamicin, and 120 (50%) to ciprofloxacin. No resistance to imipenem was detected. The 240 clinical isolates of Enterobacteriaceae that were positive for ESBL production were examined by PCR for the presence of bla CTX-M genes. Of these, 132 (55%) were positive for bla CTX-M genes, of which 124 were Esch. coli, seven were Klebsiella spp. and one was Ent. cloacae. When the 132 positive clinical isolates were further tested with CTX-M group-specific primers, 81 (61%) were positive for the CTX-M-9 group, 49 (37%) were positive for the CTX-M-1 group, and two (2%) were positive for the CTX-M-2 group (Table 2). IEF showed that CTX-M-9 group enzymes had pis of 8.0 and 8.1, the CTX-M-1 group had pis of 8.4 and 8.9, and the CTX-M-2 group had a pi of 7.8. Isolates (n = 108) belonging to the CTX-Mnegative group produced different cefotaximehydrolysing b-lactamases, with pis of that were suggestive of TEM- and SHV-type ESBLs. PCR with bla TEM -specific primers amplified a 971- bp fragment from isolates with TEM-like ESBLs, while bla SHV -specific primers amplified an 885-bp fragment from isolates with SHV-like ESBLs. Analytical IEF of the AmpC-producing isolates (n = 14) showed that all of these isolates produced enzymes with a pi of >9.0 that were inhibited by cloxacillin when using the IEF overlay technique, which is characteristic of AmpC-type cephalosporinases [28]. Three isolates

5 Pitout et al. Molecular diagnostic assay for CTX-M enzymes 295 produced an additional b-lactamase with a pi of 8.1, and two isolates produced a b-lactamase with a pi of 8.9. The duplex PCR amplified a 450-bp amplicon from these five isolates; three (pi 8.1) were positive with the CTX-M-9 group-specific primers, and two (pi 8.9) were positive with the CTX-M-1 group-specific primers. The remaining nine AmpC-producing Esch. coli isolates were negative for bla CTX-M genes. DISCUSSION The recent emergence of isolates producing CTX- M variants capable of hydrolysing ceftazidime, as well as cefotaxime, with high efficiency has rendered their phenotypic recognition extremely difficult. Among those variants are the CTX-M-15, CTX-M-16, CTX-M-19 and CTX-M-27 enzymes [4]. Of the recent clinical isolates positive for bla CTX-M genes included in the present study, 42 (32%) of 132 had increased ceftazidime MICs, confirming that the phenotypic identification of CTX-M-producing organisms is not a reliable approach. The usefulness of the PCR test, and probably other CTX-M detection methods, was also evident for AmpC-producing Esch. coli harbouring CTX-M-type b-lactamases. Molecular methods that include simple and multiplex PCR, real-time PCR, DNA sequencing, and various hybridisation-based techniques, are now used widely in academia and in reference laboratories for the detection of bacteria producing ESBLs [29]. Molecular methods have advantages over phenotypic tests in that they accurately and rapidly detect resistant genes and, by defining the precise genetic basis of the resistance mechanism, provide information valuable for reaching decisions concerning the early introduction of targeted infection control practices [30]. Molecular detection of antibiotic resistance has the potential to impact directly on patient care, but the majority of in-house tests lack proper verification and validation for routine use in a clinical laboratory [27]. The PCR described in the present study has been validated for the simultaneous detection of the different groups of CTX-M enzymes, takes c. 6 h to complete, and can be performed by any microbiologist with some molecular experience. The technique is therefore suitable for use in clinical or reference laboratories to screen, track and monitor the spread of large numbers of organisms producing CTX-M enzymes from the community and hospital settings in real-time. All positive isolates can then be further divided into the five CTX-M phylogenetic groups using multiplex PCR [20] or group-specific primers [26]. This will ensure the early recognition of an outbreak involving organisms producing CTX-M enzymes and the timely introduction of appropriate infection control procedures. The majority (55%) of ESBL-producing Enterobacteriaceae isolated recently from the CHR was positive for bla CTXM enzymes, which emphasises the current emergence and spread of CTX-M enzymes among Enterobacteriaceae in this region. It is hoped that the present study will provide a benchmark for the verification and validation of new in-house diagnostic molecular assays before their introduction into clinical microbiology laboratories. ACKNOWLEDGEMENTS This work was supported by a grant from the University of Calgary Dean s Starter Grant (# ) and Calgary Laboratory Services. We thank T. Ross for her technical support of this study, R. Bonnet for providing the control strains CF1, CF2, VER-1, Rio3, Rio4 and Rio6, M. Gniadkowski for Cfr and Eco , P. Bradford for 34, F. Navaro for 785D, R. Canton for Ec , L. Siu for BM4493, P. Courvalin for KpnBr4493, M. Mulvey for N and ESBL530, and E. Smith Moland for CT1, S1, S2, T1, T4, T5, A1, A2, C1 and C2. LP is a researcher from the INSERM, Paris, France. REFERENCES 1. Bradford PA. Extended-spectrum b-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: Paterson DL, Bonomo RA. Extended-spectrum b-lactamases: a clinical update. Clin Microbiol Rev 2005; 18: Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Enterobacteriaceae producing extended-spectrum b-lactamases (ESBLs) in the community. J Antimicrob Chemother 2005; 56: Bonnet R. Growing group of extended-spectrum b-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004; 48: Mulvey MR, Bryce E, Boyd D et al. Ambler class A extended-spectrum b-lactamase-producing Escherichia coli and Klebsiella spp. in Canadian hospitals. Antimicrob Agents Chemother 2004; 48: Pitout JD, Hanson ND, Church DL, Laupland KB. Population-based laboratory surveillance for Escherichia coli producing extended-spectrum b-lactamases: importance of community isolates with blactx-m genes. Clin Infect Dis 2004; 38:

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