Download Multiplex PCR for rapid detection of genes

Journal of Antimicrobial Chemotherapy (2007) 59, 321– 322
doi:10.1093/jac/dkl481
Advance Access publication 21 December 2006
Multiplex PCR for rapid detection of genes encoding
acquired metallo-b-lactamases
Matthew J. Ellington*†, James Kistler, David M. Livermore
and Neil Woodford
Antibiotic Resistance Monitoring and Reference Laboratory,
Centre for Infections, Health Protection Agency, London, UK
Keywords: imipenem, carbapenems, acquired resistance, IMP, VIM
*Corresponding author. Tel: þ44-20-8327-7259; Fax:þ44-208200-7449; E-mail: [email protected]
†
Present address: Staphylococcal Reference Unit, Laboratory of
Healthcare Associated Infection, Centre for Infections, Health
Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK.
Sir,
Carbapenems are considered a last-line agent for the treatment
of serious Gram-negative sepsis. Metallo-b-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 50 -GGA ATA GAG TGG CTT
AAY TCT C-30 /Imp-R 50 -CCA AAC YAC TAS GTT ATC T-30 ;
VIM family, Vim-F 50 -GAT GGT GTT TGG TCG CAT A-30 /
Vim-R 50 -CGA ATG CGC AGC ACC AG-30 ; GIM-1, Gim-F
50 -TCG ACA CAC CTT GGT CTG AA-30 /Gim-R 50 -AAC TTC
CAA CTT TGC CAT GC-30 ; SPM-1, Spm-F 50 -AAA ATC
TGG GTA CGC AAA CG-30 /Spm-R 50 -ACA TTA TCC GCT
GGA ACA GG-30 ; Sim-1, Sim-F 50 -TAC AAG GGA TTC GGC
ATC G-30 /Sim-R 50 -TAA TGG CCT GTT CCC ATG TG-30 .
DNA template was prepared by emulsifying 5 colonies in 100 mL
of PCR grade water and adding 2 mL to the PCR reaction mixture
prior to thermal cycling. The cycling conditions were: initial
DNA release and denaturation at 948C for 5 min, followed by 36
cycles of 948C for 30 s, 528C for 40 s and 728C for 50 s, followed
by a single, final, elongation step at 728C 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.
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.
Figure 1. Multiplex PCR assay for genes encoding
metallo-b-lactamases. Amplifications were from control strains.
acquired
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Correspondence
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 bacteria are a potentially grave threat to modern
intensive-care medicine. Rapid detection and good infection
control are needed to reduce their impact. This simple assay will
assist in monitoring their emergence and spread.
Acknowledgements
For providing the VIM-7-, SIM-1- and SPM-1-producing control
isolates, respectively, we would like to thank Dr Mark
A. Toleman (Bristol, UK), Professor Kyungwon Lee (Seoul,
Korea) and Dr Ana Gales (São Paulo, Brazil).
Transparency declarations
None to declare.
References
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2. Livermore DM, Woodford N. The b-lactamase threat in
Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends
Microbiol 2006; 14: 413–20.
3. Walsh TR, Toleman MA, Poirel L et al. Metallo b-lactamases: the
quiet before the storm? Clin Microbiol Rev 2005; 18: 306–25.
4. Lee K, Yum JH, Yong D et al. Novel acquired
metallo-b-lactamase gene, blaSIM-1, in a class 1 integron from
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