JAC Advance Access originally published online on November 10, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):154-155; doi:10.1093/jac/dki412
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Correspondence |
Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum ß-lactamases
Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK
* Corresponding author. Tel: +44-20-8327-7255; Fax: +44-20-8327-6264; E-mail: neil.woodford{at}hpa.org.uk
Keywords: ESBLs , antibiotic resistance , Enterobacteriaceae , CTX-M enzymes , molecular diagnostics
Sir,
CTX-M extended-spectrum ß-lactamases (ESBLs) are increasingly prevalent worldwide among Escherichia coli and Klebsiella spp., including in the UK, where over half of all microbiology laboratories have encountered producers.1,2 More than 40 CTX-M ß-lactamases have been described and are divided into five phylogenetic groups, with different groups prevalent in different countries.3 However, characterization of the blaCTX-M alleles present in clinical isolates is time-consuming, typically requiring multiple PCRs with universal and then group-specific primers.1,2,4 To facilitate monitoring, we sought to develop a multiplex assay to differentiate alleles belonging to these five phylogenetic groups.
The sequences of reference blaCTX-M alleles (source: http://www.lahey.org/studies/) were aligned, and group-specific regions were identified. Primers specific for alleles encoding enzymes belonging to groups 1, 2 and 9 were designed in silico using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), whereas those for blaCTX-M-25/-26 and for blaCTX-M-8/-40 were developed and optimized empirically. Primer pairs and predicted amplicon sizes were: group 1, 5'-AAA AAT CAC TGC GCC AGT TC and 5'-AGC TTA TTC ATC GCC ACG TT (415 bp); group 2, 5'- CGA CGC TAC CCC TGC TAT T and 5'-CCA GCG TCA GAT TTT TCA GG (552 bp); group 9, 5'-CAA AGA GAG TGC AAC GGA TG and 5'-ATT GGA AAG CGT TCA TCA CC (205 bp). Fragments of alleles encoding enzymes of groups 8 (666 bp) and 25 (327 bp) were amplified with two specific forward primers and a shared reverse primer: 5'-TCG CGT TAA GCG GAT GAT GC (group 8 forward); 5'-GCA CGA TGA CAT TCG GG (group 25 forward); and 5'-AAC CCA CGA TGT GGG TAG C (groups 8/25 reverse).
Amplification conditions were: initial denaturation at 94°C for 5 min; 30 cycles of 94°C for 25 s, 52°C for 40 s and 72°C for 50 s; and a final elongation at 72°C for 6 min. The primer pairs were evaluated separately against 14 control strains and then in a multiplex format. The controls were shown previously by sequencing to produce CTX-M-15 (n = 6), CTX-M-14 (n =2) and one producer each of CTX-M-2, -8, -9, -25, -26 and -40. The multiplexed combination of primers amplified blaCTX-M alleles from each of the control strains. Furthermore, these were assigned to their correct phylogenetic groups, as defined by the predicted sizes of amplicons (Figure 1).
|
The assay was then used to screen 633 isolates of Enterobacteriaceae, which had been submitted to the reference service for confirmation of ESBL production. In all cases interpretative reading of the resulting antibiograms suggested the presence of a CTX-M enzyme, i.e. the MIC of cefotaxime was
4-fold greater than the MIC of ceftazidime, with both MICs being significantly reduced in the presence of clavulanic acid. Alleles encoding group 1 CTX-M enzymes were found in 547 (86.4%) of the 633 isolates, comprising 429 E. coli, 114 Klebsiella spp., 3 Enterobacter spp. and 1 Morganella sp. Group 9 enzymes were produced by 81 (12.8%) isolates, comprising 74 E. coli, 6 Klebsiella spp. and 1 Enterobacter sp. The remaining isolates included: three E. coli that produced group 2 CTX-M enzymes; one E. coli that produced a group 8 enzyme; and one E. coli isolate that contained alleles encoding both a group 1 and a group 9 enzyme. No producers of group 25 CTX-M enzymes were detected. The predominance of group 1 enzymes, as seen here, has been reported previously among CTX-M producers in the UK.1 Of particular note, 113 (26%) group 1-producing E. coli isolates belonged to ‘strain A’, an epidemic UK CTX-M-15-producing strain as defined by its PFGE banding pattern and distinctive molecular characteristics.1 Such isolates were referred from 26 UK laboratories, indicating further spread of strain A since its original description.1 To our knowledge, this is also the first report of group 2 CTX-M enzymes in the UK; the isolates were from three hospitals and it is possible that they represent separate introductions. Group 2 are the most prevalent CTX-M enzymes in parts of South America and in Israel;3 group 9 enzymes are prevalent in Spain.5
In summary, we have developed a multiplex PCR assay that was able to detect and distinguish alleles encoding CTX-M enzymes belonging to all five phylogenetic groups. CTX-M ESBLs are recognized worldwide as an increasingly serious public health concern.3 Simple PCR assays, as described here and by others,6 will assist in monitoring their emergence and dissemination.
Transparency declarations
No declarations were made by the authors of this paper.
Acknowledgements
We wish to thank Vicky Ensor, Peter Hawkey, Katie Hopkins and Patrice Nordmann for providing some of the control strains included in this study. Part of this work was presented at the 15th ECCMID, Copenhagen, April 2005.
References
1.
Woodford N, Ward ME, Kaufmann ME et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum ß-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735–43.
2.
Munday CJ, Whitehead GM, Todd NJ et al. Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum ß-lactamases in York, UK. J Antimicrob Chemother 2004; 54: 628–33.
3.
Bonnet R. Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004; 48: 1–14.
4. Saladin M, Cao VT, Lambert T et al. Diversity of CTX-M ß-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 2002; 209: 161–8.[Web of Science][Medline]
5.
Hernandez JR, Martinez-Martinez L, Canton R et al. Nationwide study of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum ß-lactamases in Spain. Antimicrob Agents Chemother 2005; 49: 2122–5.
6.
Pitout JDD, Hossain A, Hanson ND. Phenotypic and molecular detection of CTX-M-ß-lactamases produced by Escherichia coli and Klebsiella spp. J Clin Microbiol 2004; 42: 5715–21.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Oxacelay, A. Ergani, T. Naas, and P. Nordmann Rapid detection of CTX-M-producing Enterobacteriaceae in urine samples J. Antimicrob. Chemother., November 1, 2009; 64(5): 986 - 989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Hrabak, J. Empel, T. Bergerova, K. Fajfrlik, P. Urbaskova, I. Kern-Zdanowicz, W. Hryniewicz, and M. Gniadkowski International Clones of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum {beta}-Lactamases in a Czech Hospital J. Clin. Microbiol., October 1, 2009; 47(10): 3353 - 3357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Doumith, M. J. Ellington, D. M. Livermore, and N. Woodford Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK J. Antimicrob. Chemother., April 1, 2009; 63(4): 659 - 667. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nagano, N. Nagano, J.-i. Wachino, K. Ishikawa, and Y. Arakawa Novel Chimeric {beta}-Lactamase CTX-M-64, a Hybrid of CTX-M-15-Like and CTX-M-14 {beta}-Lactamases, Found in a Shigella sonnei Strain Resistant to Various Oxyimino-Cephalosporins, Including Ceftazidime Antimicrob. Agents Chemother., January 1, 2009; 53(1): 69 - 74. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Mesko Meglic, S. Koren, M. F. I. Palepou, E. Karisik, D. M. Livermore, R. Pike, A. Andlovic, S. Jeverica, V. Krizan-Hergouth, M. Muller-Premru, et al. Nationwide Survey of CTX-M-Type Extended-Spectrum {beta}-Lactamases among Klebsiella pneumoniae Isolates in Slovenian Hospitals Antimicrob. Agents Chemother., January 1, 2009; 53(1): 287 - 291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zong, S. R. Partridge, L. Thomas, and J. R. Iredell Dominance of blaCTX-M within an Australian Extended-Spectrum {beta}-Lactamase Gene Pool Antimicrob. Agents Chemother., November 1, 2008; 52(11): 4198 - 4202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Reynolds, R. Hope, L. Williams, and on behalf of the BSAC Working Parties on Resistanc Survey, laboratory and statistical methods for the BSAC Resistance Surveillance Programmes J. Antimicrob. Chemother., November 1, 2008; 62(suppl_2): ii15 - ii28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Livermore, R. Hope, G. Brick, M. Lillie, R. Reynolds, and on behalf of the BSAC Working Parties on Resistanc Non-susceptibility trends among Enterobacteriaceae from bacteraemias in the UK and Ireland, 2001-06 J. Antimicrob. Chemother., November 1, 2008; 62(suppl_2): ii41 - ii54. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Valverde, F. Grill, T. M. Coque, V. Pintado, F. Baquero, R. Canton, and J. Cobo High Rate of Intestinal Colonization with Extended-Spectrum-{beta}-Lactamase-Producing Organisms in Household Contacts of Infected Community Patients J. Clin. Microbiol., August 1, 2008; 46(8): 2796 - 2799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Aschbacher, M. Doumith, D. M. Livermore, C. Larcher, and N. Woodford Linkage of acquired quinolone resistance (qnrS1) and metallo-{beta}-lactamase (blaVIM-1) genes in multiple species of Enterobacteriaceae from Bolzano, Italy J. Antimicrob. Chemother., March 1, 2008; 61(3): 515 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Warren, V. M. Ensor, P. O'Neill, V. Butler, J. Taylor, K. Nye, M. Harvey, D. M. Livermore, N. Woodford, and P. M. Hawkey Imported chicken meat as a potential source of quinolone-resistant Escherichia coli producing extended-spectrum {beta}-lactamases in the UK J. Antimicrob. Chemother., March 1, 2008; 61(3): 504 - 508. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Valverde, T. M. Coque, L. Garcia-San Miguel, F. Baquero, and R. Canton Complex molecular epidemiology of extended-spectrum -lactamases in Klebsiella pneumoniae: a long-term perspective from a single institution in Madrid J. Antimicrob. Chemother., January 1, 2008; 61(1): 64 - 72. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Karisik, M. J. Ellington, D. M. Livermore, and N. Woodford Virulence factors in Escherichia coli with CTX-M-15 and other extended-spectrum -lactamases in the UK J. Antimicrob. Chemother., January 1, 2008; 61(1): 54 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Carattoli, A. Garcia-Fernandez, P. Varesi, D. Fortini, S. Gerardi, A. Penni, C. Mancini, and A. Giordano Molecular Epidemiology of Escherichia coli Producing Extended-Spectrum -Lactamases Isolated in Rome, Italy J. Clin. Microbiol., January 1, 2008; 46(1): 103 - 108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Machado, J. Ferreira, A. Novais, L. Peixe, R. Canton, F. Baquero, and T. M. Coque Preservation of Integron Types among Enterobacteriaceae Producing Extended-Spectrum {beta}-Lactamases in a Spanish Hospital over a 15-Year Period (1988 to 2003) Antimicrob. Agents Chemother., June 1, 2007; 51(6): 2201 - 2204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Xu, J. Evans, T. Ling, K. Nye, and P. Hawkey Rapid Genotyping of CTX-M Extended-Spectrum {beta}-Lactamases by Denaturing High-Performance Liquid Chromatography Antimicrob. Agents Chemother., April 1, 2007; 51(4): 1446 - 1454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Wiegand, H. K. Geiss, D. Mack, E. Sturenburg, and H. Seifert Detection of Extended-Spectrum Beta-Lactamases among Enterobacteriaceae by Use of Semiautomated Microbiology Systems and Manual Detection Procedures J. Clin. Microbiol., April 1, 2007; 45(4): 1167 - 1174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Ensor, D. M. Livermore, and P. M. Hawkey A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum {beta}-lactamases J. Antimicrob. Chemother., March 1, 2007; 59(3): 387 - 395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Novais, R. Canton, R. Moreira, L. Peixe, F. Baquero, and T. M. Coque Emergence and Dissemination of Enterobacteriaceae Isolates Producing CTX-M-1-Like Enzymes in Spain Are Associated with IncFII (CTX-M-15) and Broad-Host-Range (CTX-M-1, -3, and -32) Plasmids Antimicrob. Agents Chemother., February 1, 2007; 51(2): 796 - 799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Naas, C. Oxacelay, and P. Nordmann Identification of CTX-M-Type Extended-Spectrum-{beta}-Lactamase Genes Using Real-Time PCR and Pyrosequencing Antimicrob. Agents Chemother., January 1, 2007; 51(1): 223 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Woodford, S. Reddy, E. J. Fagan, R. L. R. Hill, K. L. Hopkins, M. E. Kaufmann, J. Kistler, M.-F. I. Palepou, R. Pike, M. E. Ward, et al. Wide geographic spread of diverse acquired AmpC {beta}-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland J. Antimicrob. Chemother., January 1, 2007; 59(1): 102 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. I. Birkett, H. A. Ludlam, N. Woodford, D. F. J. Brown, N. M. Brown, M. T. M. Roberts, N. Milner, and M. D. Curran Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum {beta}-lactamases J. Med. Microbiol., January 1, 2007; 56(1): 52 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Karisik, M. J. Ellington, R. Pike, R. E. Warren, D. M. Livermore, and N. Woodford Molecular characterization of plasmids encoding CTX-M-15 {beta}-lactamases from Escherichia coli strains in the United Kingdom J. Antimicrob. Chemother., September 1, 2006; 58(3): 665 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Livermore, R. Hope, E. J. Fagan, M. Warner, N. Woodford, and N. Potz Activity of temocillin against prevalent ESBL- and AmpC-producing Enterobacteriaceae from south-east England J. Antimicrob. Chemother., May 1, 2006; 57(5): 1012 - 1014. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||