JAC Advance Access published online on September 26, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn400
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Original research |
Detection of Pseudomonas aeruginosa isolates producing VEB-type extended-spectrum β-lactamases in the United Kingdom
1 Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK 2 Department of Microbiology, Wrightington, Wigan and Leigh NHS Trust, Wigan, UK 3 Whiston Hospital, St Helens and Knowsley Teaching Hospitals NHS Trust, Prescot, Merseyside, UK 4 Department of Microbiology, The Great Western Hospital, Swindon, UK 5 Department of Medical Microbiology, Salisbury NHS Foundation Trust, Salisbury, UK
* Corresponding author. Tel: +44-20-8327-7255; Fax: +44-20-8327-6264; E-mail: neil.woodford{at}hpa.org.uk
Received 10 July 2008; returned 22 August 2008; revised 27 August 2008; accepted 29 August 2008
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Abstract |
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Objectives: The aim of this study was to investigate the presence of VEB enzymes among Pseudomonas spp. referred to the UK's national reference laboratory and with phenotypic evidence of extended-spectrum β-lactamase (ESBL) production.
Methods: Antibiograms were analysed for Pseudomonas spp. referred from November 2003 to November 2007. Isolates with 
4-fold ceftazidime/clavulanate synergy were screened for blaVEB alleles. Genes encoding metallo-β-lactamases (blaMBL) were sought in isolates with positive imipenem/EDTA synergy tests. Selected PCR products were sequenced. PFGE of SpeI-digested genomic DNA was used to compare isolates.
Results: Forty-nine (3.7%) of 1338 Pseudomonas spp. were considered potential ESBL producers; 40 were recovered for molecular testing. blaVEB alleles were detected in 32 Pseudomonas aeruginosa isolates, comprising diverse PFGE types, from 12 UK hospitals and 1 in India. One UK centre referred 15 isolates with VEB-1 enzyme; these were serotype O15, representing a single PFGE-defined strain that also produced VIM-10 metallo-carbapenemase. This strain was resistant to all β-lactams, aminoglycosides and ciprofloxacin, remaining susceptible only to colistin (MICs 
1 mg/L). Two other P. aeruginosa isolates co-producing both VEB and VIM enzymes were received from two other UK hospitals; one isolate represented inter-hospital spread of the O15 strain and the second was distinct.
Conclusions: VEB enzymes have not been reported previously in the UK, but were produced by 80% of Pseudomonas spp. with phenotypic evidence of ESBL production. They co-existed with VIM carbapenemases in two strains, with one responsible for a major hospital outbreak. The incidence of ESBLs may be underestimated in Pseudomonas because ESBL phenotypes can be masked by other β-lactam resistance mechanisms.
Key Words: ESBL , VIM metallo-carbapenemase , international clone , hospital outbreak
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Introduction |
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The rise to dominance of CTX-M-type extended-spectrum β-lactamases (ESBLs) among members of the Enterobacteriaceae, particularly Escherichia coli, is one of the most dramatic shifts in resistance epidemiology of the early 21st century,1,2 supplementing the problems already caused by TEM- and SHV-derived ESBLs. In addition, a variety of ‘minor’ class A ESBLs have been described,3 with the VEB enzymes representing an increasingly seen group.3 VEB-1 enzyme was first characterized in an E. coli isolate from Vietnam in 1996, with four further sequence variants since detected in widely scattered countries, in members of the Enterobacteriaceae or in non-fermenting genera, particularly Pseudomonas spp.3
Cephalosporin/clavulanate ESBL tests are widely agreed to have, in general, poor sensitivity for Pseudomonas spp. However, VEB enzymes are well inhibited by clavulanate, and significant reductions in ceftazidime MICs have been reported for VEB ESBL-producing Pseudomonas spp.3,4 For logistic reasons, the HPA's Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL) determines the susceptibilities of Pseudomonas spp. in parallel with Enterobacteriaceae by agar dilution, including on cephalosporin/clavulanate plates. We noted that some Pseudomonas isolates showed ceftazidime/clavulanate synergy in these tests and investigated the presence of blaVEB genes among these.
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Bacterial isolates
Antibiograms were reviewed for all isolates of Pseudomonas spp. referred to ARMRL from November 2003 to November 2007. These had been determined by agar dilution methodology. MICs were interpreted using harmonized European Committee for Antimicrobial Susceptibility Testing (EUCAST)/BSAC (v.6) breakpoints. Isolates were suspected to be ESBL producers if they showed a 4-fold reduction in ceftazidime MIC in the presence of 4 mg/L clavulanate. These isolates were recovered from storage, either at –70°C or at room temperature, for molecular investigation.
Isolates were screened by PCR for blaVEB alleles with primers VEBcas-F and VEBcas-B.5 In addition, blaMBL alleles (encoding enzymes of the IMP, VIM, SPM, GIM and SIM groups) were sought by multiplex PCR, as described previously,6 in isolates that also showed positive imipenem/EDTA synergy tests. Selected PCR products were sequenced using dye-terminator chemistry on a CEQ8000 Genetic Analyser (Beckman Coulter, High Wycombe, UK). Sequences were compared and aligned with reference sequences using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and CLUSTAL W (http://www.ebi.ac.uk/clustalw).
PFGE of SpeI-digested genomic DNA was employed to examine the relatedness of selected isolates, with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) used to determine relatedness between isolates.
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MIC analysis and blaVEB screening
Ceftazidime and ceftazidime/clavulanic acid MICs were reviewed for 1338 Pseudomonas spp. isolates referred during the 4 year period. Almost half (630, 47%) were resistant to ceftazidime (MIC > 8 mg/L), which emphasizes the bias towards resistance among isolates submitted to the reference laboratory. For comparison, the BSAC bacteraemia surveillance programme detected ceftazidime resistance in only 1.5% of the Pseudomonas spp. isolates tested during 2006 (http://www.bsacsurv.org/mrsweb/bacteraemia).
Clavulanic acid reduced the ceftazidime MICs for 49 (3.7%) isolates by 4-fold, and these were considered to be potential ESBL producers; all were highly resistant to ceftazidime (MICs 64 mg/L). Forty of these potential ESBL producers were recovered for molecular testing, and a blaVEB allele was detected in 32 (80%) of these, referred from 12 UK hospitals and from 1 in India. All of the VEB ESBL-producing isolates were confirmed to be P. aeruginosa.
Recognition of a hospital outbreak: co-production of VEB ESBL and VIM metallo-carbapenemase
Of the 32 P. aeruginosa isolates with VEB ESBLs, 15 were referred from one UK centre (hospital A). These isolates had been recovered from patients on an intensive care unit and were referred in 2003 (1 isolate), 2006 (3 isolates) and 2007 (11 isolates). All belonged to serotype O15 and represented a single PFGE-defined strain (Figure 1). These isolates were multiresistant to carbapenems and other β-lactams, aminoglycosides and ciprofloxacin, but remained susceptible to colistin (Table 1). They showed marked synergy between imipenem and EDTA (Table 1) and were positive for a blaVIM allele in addition to blaVEB. Sequencing of the PCR products from a representative of this strain identified blaVEB-1a and blaVIM-10. The blaVEB-1a allele, which was originally associated with India,7 encodes classic VEB-1 ESBL, but has an amino acid substitution in the signal peptide.8 The VIM-10 metallo-carbapenemase is a variant that was originally identified in the UK (GenBank no. AY524989 [GenBank] ).
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A review of records identified the likely ‘index case’ in hospital A's outbreak. This patient had been transferred to the UK in May 2000 from a hospital in India. A P. aeruginosa isolate from this patient was subsequently recovered from storage and referred for testing. It also belonged to serotype O15, was indistinguishable from the outbreak strain by PFGE (Figure 1) and was positive for blaVEB. However, it lacked blaVIM and was susceptible to carbapenems (Table 1).
Two serotype O15 P. aeruginosa isolates from other UK hospitals clustered by PFGE with the strain from hospital A (Figure 1). One of these, from hospital B, co-produced VEB and VIM enzymes and was linked with transfer of a patient from hospital A; the other, from hospital C, produced VEB enzyme only. The latter patient had been repatriated to the UK from Thailand in 2007 and had no known links with hospital A or B.
Only one other P. aeruginosa isolate co-producing both VEB and VIM β-lactamases was detected (from hospital D). It represented a distinct strain (Figure 1), and a second isolate of this strain (from the same patient) produced only VEB ESBL. The remaining isolates, from eight UK laboratories and one in India, had blaVEB alone, and there was no evidence of strain spread between patients or centres.
In summary, although VEB ESBLs have not been reported previously in the UK, they proved to be the predominant ESBLs among Pseudomonas spp., which showed clear phenotypic evidence of ESBL production, present in 80% (32/40) of such isolates. However, blaVEB alleles were not detected in eight potential ESBL-producing Pseudomonas isolates, six of which showed 16-fold reduction of ceftazidime MICs by 4 mg/L clavulanate. Further work is required to define the ESBL genes responsible for these phenotypes.
We have demonstrated that VEB ESBLs are widespread in the UK, with producers referred from 12 different bacteriology laboratories. Worryingly, VEB ESBLs were found to co-exist with VIM metallo-carbapenemases in two strains of P. aeruginosa, and we identified one such strain that had caused a prolonged hospital outbreak. In this case, molecular data supported a scenario, in which (i) an O15 strain of P. aeruginosa producing VEB-1 ESBL was imported from India in 2000 to hospital A where (ii) it acquired VIM-10 metallo-carbapenemase from an unknown source and then spread to cause the outbreak. Our investigation further suggested that this serotype O15, VEB-positive P. aeruginosa strain may be widespread in the Far East, as a second representative (VIM-negative) was imported into the UK in 2007 from Thailand.
Unless molecular screening is applied to all ceftazidime-resistant isolates, the incidence of VEB and other ESBLs will be underestimated in Pseudomonas spp., because ESBL phenotypes can be masked by the genus' other β-lactam resistance mechanisms. However, such screening is an unrealistic proposition even in reference laboratories. Fortunately, the presence of ESBLs in Pseudomonas spp. rarely causes specific therapeutic dilemmas, and the genus seems not to be a major reservoir of ESBL genes for members of the Enterobacteriaceae, where CTX-M-type enzymes dominate.1
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No external funding was provided for this work.
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N. W. and D. M. L. have received research grants and speaking invites from various pharmaceutical companies. D. M. L. has a diversified share portfolio, including holdings in pharmaceutical companies. None of these poses a conflict of interest with this work. Other authors: none to declare.
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References |
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