Multiplex PCR for rapid detection of genes encoding acquired metallo- -lactamases

Abstract

Sir, Carbapenems are considered a last-line agent for the treatment of serious Gram-negative sepsis. Metallo-β-lactamases (MBLs) hydrolyse carbapenems efficiently and, when present, can undermine carbapenem therapy.1 Acquired MBLs have been reported mainly in clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp., sometimes from major clonal outbreaks, as well as in other non-fermenters; they have also been reported, less commonly, in members of the Enterobacteriaceae.2 The acquired MBLs so far described belong to five different families3,4 with multiple variants of the VIM and IMP families and single members of the SPM, GIM and SIM families. Except in Brazil, the IMP and VIM types are the most widely reported.2 The genes for all these MBLs may be carried on mobile genetic elements, or may become chromosomally integrated.3 Given the possibility of horizontal transfer within and between species and genera, it is crucial to have tools available to monitor their dissemination to aid infection control. We sought to develop a multiplex PCR assay to detect and differentiate each of these five families of acquired MBL genes in a single reaction. Conserved regions of all available, GenBank-deposited (detailed at: http://www.lahey.org/Studies/other.asp#table1), blaIMP and blaVIM alleles were identified in clustal multiple alignments. Five primer pairs, specific for each family of acquired MBLs, were designed to amplify fragments of 188 bp (IMP), 390 bp (VIM), 271 bp (SPM-1), 477 bp (GIM-1) and 570 bp (SIM-1); these were then evaluated separately and in a multiplex format with all 10 primers. The primer pairs were: IMP family, Imp-F 5′-GGA ATA GAG TGG CTT AAY TCT C-3′/Imp-R 5′-CCA AAC YAC TAS GTT ATC T-3′; VIM family, Vim-F 5′-GAT GGT GTT TGG TCG CAT A-3′/Vim-R 5′-CGA ATG CGC AGC ACC AG-3′; GIM-1, Gim-F 5′-TCG ACA CAC CTT GGT CTG AA-3′/Gim-R 5′-AAC TTC CAA CTT TGC CAT GC-3′; SPM-1, Spm-F 5′-AAA ATC TGG GTA CGC AAA CG-3′/Spm-R 5′-ACA TTA TCC GCT GGA ACA GG-3′; Sim-1, Sim-F 5′-TAC AAG GGA TTC GGC ATC G-3′/Sim-R 5′-TAA TGG CCT GTT CCC ATG TG-3′. DNA template was prepared by emulsifying 5 colonies in 100 µL of PCR grade water and adding 2 µL to the PCR reaction mixture prior to thermal cycling. The cycling conditions were: initial DNA release and denaturation at 94°C for 5 min, followed by 36 cycles of 94°C for 30 s, 52°C for 40 s and 72°C for 50 s, followed by a single, final, elongation step at 72°C for 5 min. The assay was tested with 11 reference strains known, based on DNA sequencing, to produce IMP-1, -2, -4, and -7, -12, VIM-1, -2 and -7, SIM-1, GIM-1 and SPM-1 enzymes. All the host organisms were P. aeruginosa, with the exception of Acinetobacter spp. isolates producing IMP-2 and IMP-4, and Acinetobacter baumannii producing SIM-1. The MBL types assigned by this assay were consistent with the MBL alleles previously found by sequencing in all these 11 reference organisms, with amplicons matching the predicted sizes (Figure 1). To confirm MBL expression phenotypically in these strains, imipenem MICs were determined by agar dilution according to BSAC methodology (http://www.bsac.org.uk) with and without the addition of 320 mg/L EDTA. In all cases >4-fold potentiation was seen, consistent with MBL activity.1 Multiplex PCR assay for genes encoding acquired metallo-β-lactamases. Amplifications were from control strains. We also tested 60 UK clinical isolates referred to the UK national Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL) between 2000 and April 2006. Some had been shown by DNA sequencing to carry either blaIMP-1, -13, blaVIM-1, -4, -9 or -10; others had unsequenced blaIMP or blaVIM genes as detected in single PCR tests. The multiplex PCR assay results agreed with the previous single PCR assay results, showing that of the 60 clinical isolates, 11 had blaIMP alleles while 49 had blaVIM alleles. Those organisms with blaIMPs comprised eight Pseudomonas spp., two Klebsiella spp. and one Acinetobacter junii; those with blaVIM comprised 48 Pseudomonas spp. and one Klebsiella sp. Based on their DNA and protein sequences, VIM MBLs can be split into three subgroups; VIM-1-like, VIM-2-like and VIM-7, and representatives of each of these were detected in this assay. IMP MBLs have more disparate DNA sequences than VIM-types, but this assay detected genes for IMP-1, -2, -4, -7, -12 and -13 which diverge by as much as 16%. Only blaIMP-3 and blaIMP-16 show additional variance within the primer binding regions to the blaIMP genes detected here, each having an additional single base change compared with the forward primer. No isolates with SPM-1, GIM-1 or SIM-1 enzymes have yet been identified in the UK, but MBL producers would easily be detected by this assay, as shown with the reference strains. In summary, this multiplex PCR detected and distinguished alleles encoding five different families of acquired MBLs and found either IMP or VIM alleles in all 60 UK isolates tested. MBL-producing bacteri