JAC Advance Access originally published online on January 25, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):387-395; doi:10.1093/jac/dkl505
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A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum ß-lactamases
1 West Midlands Health Protection Agency, Heart of England NHS Foundation Trust, Bordesley Green, Birmingham B9 5SS, UK 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, UK 3 Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, Colindale, London NW9 5EQ, UK
* Corresponding author. Tel: +44-121-424-1240; Fax: +44-121-772-6229; E-mail: vicki.ensor{at}heartofengland.nhs.uk
Received 26 October 2006; returned 13 November 2006; revised 16 November 2006; accepted 17 November 2006
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Abstract |
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Objectives: To develop a reverse-line hybridization assay to identify CTX-M genotypes, potentially useful for large-scale investigation of surveillance collections.
Methods: Isolates carrying previously characterized blaCTX-M genes were used to develop the method. In addition, 334 isolates from five separate surveys were used to validate the method. CTX-M group was known from an independent multiplex PCR for 122 isolates and genotype was confirmed for 80 isolates by DNA sequencing. A multiplex PCR was designed to amplify a genotype-specific region within the blaCTX-M open-reading frame. Oligonucleotides were designed to hybridize to regions within each amplicon, covering mutations that distinguish among blaCTX-M genotypes.
Results: CTX-M phylogenetic groups were identified by the multiplex PCR with 100% concordance. The reverse-line hybridization assay specifically identified commonly-reported variants within these groups (98.7% concordance).
Conclusions: The hybridization method enabled precise identification of CTX-M genes, rather than just to group level, without the need for DNA sequencing. In its present format, the method enables 43 isolates to be processed per membrane, giving results within one working day. It is a useful tool for the epidemiological investigation of blaCTX-M genes among survey collections of Enterobacteriaceae.
Keywords: ESBLs , genotyping , multiplex PCR , molecular epidemiology
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Introduction |
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Over the last two decades, reports of Enterobacteriaceae producing extended-spectrum ß-lactamases (ESBLs) have been increasing, from every inhabited continent. Most recently this increase is attributed to the rapid and wide dissemination of CTX-M-type ESBLs.1,2 These are plasmid-borne Ambler class A enzymes that are divided into five major phylogenetic groups based on amino acid identities (groups 1, 2, 8, 25 and 9), with the predominant groups varying geographically.1,3 The recent proliferation of isolates with CTX-M enzymes is of serious public health concern. Infections caused by Enterobacteriaceae with CTX-M enzymes have been reported in community patients with little or no hospital contact.4,5 Producers are often resistant to non-ß-lactam antibiotics, complicating treatment and driving the use of carbapenems. Monitoring of the prevalence and further evolution of CTX-M enzymes is therefore of utmost importance. At present, ESBL production is usually detected by phenotypic methods based on cephalosporin/clavulanate synergy (e.g. http://www.bsac.org.uk/_db/_documents/qsop51i1.1.pdf). Identification of precise ESBL genotypes requires molecular methods and is beyond the remit of most clinical diagnostic laboratories; rather, such work is performed in reference or research laboratories. These use DNA sequencing as the gold standard for identifying blaCTX-M genotypes, but the cost per test, the requirement for specialist equipment, and the time needed to analyse sequence data preclude use on large collections from surveillance studies. Such surveys often report only the presence of blaCTX-M genes or identify these genes to group level, based on single PCR,6 multiplex PCR7–9 or real-time PCR.10
DNA oligonucleotide arrays potentially provide a route to more precise identification of genes. Using a small panel of oligonucleotides in a macroarray format, Morcillo et al.11 developed a low-cost reverse-line hybridization (RLH) assay to detect rifampicin resistance in Mycobacterium tuberculosis. The principle of an RLH assay is that a panel of synthetic oligonucleotides incorporating mutation(s) of interest is immobilized on a membrane. Target DNA is amplified by PCR and applied to the membrane at right angles to the synthetic oligonucleotides, so that it can interact with each oligonucleotide. After hybridizing and washing under stringent conditions, specific hybrids are detected.
We sought to develop a method to identify precise CTX-M genotypes without DNA sequencing, based on reverse-line hybridization (RLH) macroarray technology previously described.11
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Control strains
Sixteen sequence-confirmed control strains were used to develop and optimize the assay. These produced the following ß-lactamases: group 1: CTX-M-1, -3, -12, -15 and -32; group 2: CTX-M-2, -6, -7 and Toho-1; group 8: CTX-M-8, -26 and group 9: CTX-M-9, -13, -14, -17 and -19.
Design of group-specific primers for multiplex PCR
Regions within the blaCTX-M open-reading frame (ORF) that were conserved within, and specific to, each CTX-M group (1, 2, 8/25 or 9) were identified. Primers were designed within these regions, aiming for similar melting temperatures, and always ensuring that the predicted amplicons contained oligonucleotide sequences specific for the particular CTX-M group. Some of the primers were designed with degenerate bases to allow for substitution of one or two bases within the corresponding region for some group members. Primers were synthesized by MWG Biotech-AG, Ebersberg, Germany and the reverse primers were biotinylated on C6 at the 5'-termini; those containing degenerate bases were synthesized in equal ratios. Table 1 details the primer sequences, melting temperatures and annealing positions within the blaCTX-M ORF, along with the predicted amplicon sizes.
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Multiplex PCR
Template DNA was prepared from freshly-cultured control strains by touching two or three discrete colonies and suspending the collected material in 50 µL of molecular grade water, then heating at 95°C for 5 min. Each reaction consisted of 21.75 µL of Mastermix (ABgene, Epsom, UK), 0.25 µL of each primer (100 pmol) and 2.0 µL of template DNA. Optimal PCR conditions were determined to be as follows: 1 cycle at 95°C for 5 min followed by 25 cycles of 95°C for 30 s, 63°C for 30 s and 72°C for 1 min, with a final elongation at 72°C for 10 min. Amplicons were visualized using a transilluminator, after running on 2% agarose gel at 150 V for 2 h, and staining in ethidium bromide. The amplicons were then kept at 4°C prior to use. The presence of a band of the predicted size indicated carriage of blaCTX-M by the test isolate, and CTX-M phylogenetic groups were identified by the amplicon size.
Design of sequence-specific DNA oligonucleotides
A list of the recognized CTX-M enzymes was obtained from http://www.lahey.org/studies/inc_webt.asp (accessed September 2004). Phylogenetic groups were assigned by constructing a dendrogram and clustering blaCTX-M genes into their four main groups: 1, 2, 8 and 9. CTX-M-25 and -26 enzymes were counted as members of group 8 at the time of design, although they are now grouped separately. Clustal W software (version 1.86) was used to align blaCTX-M nucleotide sequences within groups. Key mutation(s) that discriminate among CTX-M genotypes within the same group were identified and short oligonucleotides (at least 15 bp in length, to ensure specificity) were designed to incorporate each key mutation. The critical base mutation within each oligonucleotide was designed to appear at least in the centre or preferably two-thirds of the way towards the 3' terminus, so that the crucial part of the oligonucleotide projected from the membrane surface and was available for hybridization. Each panel of oligonucleotides (four panels in total) was designed to have similar melting temperatures and therefore also the same optimal washing temperature. The melting temperature was estimated using a formula available on the ordering page of the MWG web site (http://www.mwg-biotech.com). BLAST 2 Sequence alignment software (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) was used to ensure oligonucleotide sequences were specific and did not cross-hybridize with other sequences within the amplified region of the blaCTX-M genes. Oligonucleotides were synthesized by MWG Biotech-AG, Ebersberg, Germany with an amino-link at the 5' terminus for covalent binding to activated carboxyl groups on the membrane (see below for details). Each oligonucleotide was assigned a two-digit identification number, the first number relating to the CTX-M group with which it was designed to be used, and the second being a unique identifier for the particular oligonucleotide. For example, oligonucleotide 1–2 was designed to be used in the identification of members of CTX-M group 1, and was the second oligonucleotide designed for that group. Some oligonucleotides were subsequently discarded and so numbering is not always sequential. Table 2 shows details of the oligonucleotide panels designed for each CTX-M group.
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Preparation of the membrane for reverse line hybridization
Biodyne C Negatively Charged Nylon Membrane with a pore size of 0.45 µm (Pall Gelman Laboratory, Portsmouth, UK) was cut to the size of a support cushion (Immunetics, Boston MA, USA). The membrane was activated by washing with 10 mL of 16% N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) solution (Sigma-Aldrich, Gillingham, UK) at room temperature for 15 min, then was rinsed with distilled water for 2 min, marked for orientation, and placed on the support cushion before being inserted into the immunoblotter (Miniblotter MN45; Immunetics).
Applying oligonucleotides to the membrane
Due to differences in optimal hybridization and washing temperatures, separate membranes were prepared for each CTX-M group-specific panel of oligonucleotides. The optimal concentration for each oligonucleotide was determined by performing a series of titration experiments. Each oligonucleotide was then diluted to its optimal concentration in 0.5 M NaHCO3, pH 8.4 (BDH, Poole, UK) and 150 µL of the diluted solution was applied to the membrane by pipetting into a lane of the immunoblotter, with care to avoid the formation of air bubbles. After 1 min, excess oligonucleotide was aspirated and the membrane deactivated by washing in 100 mM NaOH (BDH) for 8 min.
Applying amplicons to the membrane for hybridization
A membrane with appropriate group-specific oligonucleotides was selected according to the CTX-M group detected by multiplex PCR. After washing at 50°C in 0.1% SDS/2 x SSPE for 10 min, the membrane was placed on a fresh support cushion and re-inserted into the immunoblotter at 90° to its original orientation. Excess solution was aspirated from each lane. Amplicon (10 µL) was added to 150 µL of 0.1% SDS/2 x SSPE, heated at 95° C for 5 min and immediately placed on ice to ensure the amplicon remained single stranded. Each lane of the immunoblotter was then filled with 150 µL of amplicon/SDS/SSPE solution. Lanes 1 and 45 were filled with 0.1% SDS/2 x SSPE without amplicon, as was any unused lane. The immunoblotter holding the membrane was then transferred to an oven for hybridization at 50 °C for 45 min, after which excess amplicon was aspirated from each lane using a pipette.
To remove non-specific hybrids, the membrane was taken from the immunoblotter and washed twice for 10 min in 0.5% SDS/2 x SSPE at the predetermined optimal wash temperature (Table 2). It was then washed in 10 mL of 0.5% SDS/2 x SSPE containing 5 µL 500 U streptavidin–peroxidase conjugate (Roche Applied Science, Penzberg, Germany) for 30 min at 42 °C. Unbound conjugate was removed by washing twice, each for 10 min, in 0.5% SDS/2 x SSPE at 42 °C, followed by two more washes in 2 x SSPE, each for 5 min at room temperature, to remove any SDS. Hybrids were visualized by enhanced chemiluminescence (ECL, Amersham Biosciences, Amersham, UK) and exposure to a light-sensitive film (Hyperfilm ECL; Amersham Biosciences, Amersham, UK) used according to the manufacturer's instructions.
Interpretation of hybridization patterns
The expected hybridization profile for each CTX-M genotype was predicted based upon the correspondence of each CTX-M-encoding region and each oligonucleotide sequence. Optimal oligonucleotide concentration and washing temperatures were determined using a concentration/temperature titration approach, until the observed profiles for the controls of each genotype available matched the predicted patterns. For those CTX-M genotypes not available to us for testing, a profile was predicted. Table 3 lists the confirmed and the predicted hybridization profiles for all currently described members (August 2006) of each CTX-M group. Frequently reported genotypes are indicated in bold.
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Validation of the RLH method
In order to validate both the multiplex PCR and the novel RLH assay, five geographically diverse collections of oxyimino-cephalosporin-resistant Enterobacteriaceae were kindly provided by colleagues in the UK and overseas. Collection 1 (n = 122) comprised UK isolates from 2004, with blaCTX-M genes identified to group level by multiplex PCR.12 Collection 2 (n = 42) comprised isolates from two laboratories in the UK Midlands, collected in 2006 (provided by R. Warren and K. Nye). The third collection comprised 91 isolates from a North London hospital, collected in 2005 (provided by P. Kumari and J. Mandozana).13 The fourth collection of 44 isolates was isolated by a laboratory in Kuwait. These isolates were collected in 2005–06 (provided by V. Rotimi). The final collection comprised 130 isolates from India collected in 2003–04.14 Bi-directional DNA sequencing of the entire blaCTX-M ORF was used as the gold standard method to identify CTX-M genotypes present in a sample (n = 80) of the isolates confirmed to have blaCTX-M genes by the multiplex PCR. Samples for sequencing were selected to represent examples of all species from all sub-collections. All non-CTX-M-15 genotypes (n = 41) were included for sequencing as well as 39 of the CTX-M-15 genotypes, with at least 5 of these being selected from each of the 5 sub-collections.
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Multiplex PCR
After determining optimal amplification conditions, amplicons of the expected sizes were obtained from all the control strains, confirming the group specificity of the primers (Figure 1).
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Reverse-line hybridization (RLH)
After determining optimal conditions for each group, expected profiles were obtained for all control strains available for testing, enabling identification of CTX-M genotype by hybridization profile. Figure 2 illustrates the results obtained. In each film, oligonucleotides run from top to bottom, while test amplicons run from left to right.
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Validation of the RLH Assay
After optimizing both the multiplex PCR and the hybridization methods, characterization of the five survey collections (429 isolates in total) was performed. A typical example of a film of survey strains is shown in Figure 3. In total, 334 isolates were found to carry blaCTX-M as tested by multiplex PCR. These included 122 isolates that had previously been found to carry this gene by an alternative multiplex PCR method,8 with complete concordance as regards CTX-M group identified. Bi-directional DNA sequencing was used to verify the hybridization result for 80 isolates. These included CTX-M-1 (n = 1), CTX-M-2 (n = 14), CTX-M-3 (n = 2), CTX-M-8 (n = 1), CTX-M-9 (n = 7), CTX-M-14 (n = 15) and CTX-M-15 (n = 40). With one exception, the hybridization and DNA sequencing results all concurred. The exception was a strain collected in a London/South East England survey that was suspected to carry blaCTX-M-9 according to RLH, where a 9–15 profile was consistently obtained, and a weak signal was intermittently obtained with oligonucleotide 9–4, whereas sequencing revealed a cytosine for thymine substitution at base position 701 and a guanine for adenine substitution at base position 725. Using the numbering system described by Ambler et al.,15 this results in an Ala231Val and Asp240Gly substitution, respectively. This sequence has been independently confirmed (N. Woodford and E. Karisik, personal communication).
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Discussion |
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More than 50 CTX-M genotypes have now been described, although epidemiological reports demonstrate that only a few enzymes are frequent, with the predominant types varying with the country. For example, CTX-M-1 is dominant in Italy,16 while CTX-M-2 has long been endemic in Argentina17 and is now also the most common type in Israel.18 CTX-M-3 is the most frequently encountered ESBL type in Poland,19 while CTX-M-9, -10 and -14 are common in Spain.20,21 In the Far East, CTX-M-3, -9 and -14 are the predominant CTX-M types.22–25 CTX-M-15 has very rapidly become the most common CTX-M type worldwide with regard to number of reporting countries.4,24–33 It is the dominant CTX-M type in the UK and continental Europe (except Spain and Poland), and also in India.14
In order to monitor further dissemination, and to assess the impact of intervention strategies, a cost-effective and efficient molecular tool is needed to identify CTX-M genotypes rapidly and easily. We have developed a novel reverse-line hybridization assay for this purpose. This can reliably identify common CTX-M types, including CTX-M-1, -2, -3, -9, -14 and -15, without the need for DNA sequencing. In addition, we have designed oligonucleotides that should allow identification of types not available to us for testing. It must, however, be noted that the same hybridization profiles are obtained for multiple genotypes within a group. For example, CTX-M-15 and CTX-M-28 both have a 1–2, 1–3 profile and are not discriminated. These two genotypes differ in coding sequence by two nucleotides: at base positions 21 and 865, where blaCTX-M-15 has cytosine and guanine residues, respectively, and blaCTX-M-28 has thymine and adenine. To distinguish between these genotypes, two oligonucleotides were designed around the mutation at position 865 (position 21 is outside the amplified region), but unfortunately, non-specific hybrids were repeatedly obtained with both oligonucleotides despite numerous attempts at optimizing conditions. The melting temperature for each was 59°C, and was comparable ( ± 1°C) to the other oligonucleotides designed for group 1 genes. It was not possible to increase the wash temperature sufficiently to eliminate non-specific hybridization without also eliminating specific hybridization products. While this is a limitation it should be appreciated that CTX-M-15 is much more common than CTX-M-28 and that a profile of 2–2, 2–3 for a group 1 CTX-M member, on the basis of probability, identifies the presence of CTX-M-15. It is possible that new variants may be missed or misidentified due to mutations occurring in the blaCTX-M ORF either outside or within the PCR primer annealing site, or at positions not covered by the current panel of oligonucleotides. Indeed, one new allelic variant of blaCTX-M-9, with coding mutations resulting in Ala-231 
Val and Asp-240 Gly substitutions, was not easily distinguished from the classical form of the gene. The potential to miss new variants is a weakness with all oligonucleotide arrays that do not completely cover the coding region, and if the method becomes widely used, it will be necessary periodically to select a representative number of test isolates for confirmation of CTX-M genotype by DNA sequencing. During optimization of this study, we tested a panel of control strains received from other centres, with blaCTX-M genes characterized previously by DNA sequencing. However, after failing to obtain expected profiles on numerous occasions for a few of these controls, we performed DNA sequencing ourselves, and found discordant results with those found previously, with one or two key differences from the original data. Importantly, however, our sequencing concurred with the RLH results, casting doubt on the accuracy of the original sequencing data. DNA sequencing chromatograms are difficult and subjective to analyse and, unless their quality is excellent, minor errors are likely to creep into reported sequences. For this reason, we believe it essential to achieve good quality sequencing on at least two independent PCR amplification products, and perhaps even to have the sequence confirmed by an independent laboratory before submitting to GenBank or the Lahey Clinic's web site (http://www.lahey.org/studies/inc_webt.asp) before reporting an enzyme as a new variant. The fact that blaCTX-M genes occur on plasmids and in association with highly mobile elements no doubt explains, at least in part, their success in disseminating so widely and so rapidly, though the clonal success of some host strains is also a factor in the UK.1,4 In any event, close monitoring of their further spread is required. To this end we have developed a novel assay that enables identification of blaCTX-M within one working day and has the advantage of being very simple to perform. It does not require expensive equipment and, in its present format, enables up to 43 isolates to be tested in parallel. The high positive predictive and accuracy value of the multiplex PCR for group identification (100% each) and the high accuracy of the RLH compared with DNA sequencing for genotype identification (79/80, 98.8%) make this test a valuable tool for large-scale epidemiological surveys.
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V. E.: none to declare. P. M. H. is Grants Secretary for the British Society for Antimicrobial Chemotherapy but was excluded from all aspects of the grant review process during the review of this application. D. M. L.: none relevant to this study.
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Acknowledgements |
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We are grateful to L. Tzouvelakis, R. Bonnet, S. Kariuki, P. Nordmann and G. Bou and their teams for very kindly providing control isolates used in the development and validation of this work. We thank M. Warner, R. Warren, K. Nye, P. Kumari, J. Mandozana and V. Rotimi for providing some of the isolates used in the validation of this assay. This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2005. This work was financially supported by the British Society for Antimicrobial Chemotherapy, grant number GA550. We acknowledge receipt of BBSRC grant 6/JIF13209 awarded to the Functional Genomics Laboratory, University of Birmingham, UK, which supports DNA sequencing work, and are grateful to J. Moore for secretarial support.
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References |
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