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TRANSCRIPT
Dominance of international ‘high-risk clones’ among Metallo- -β
Lactamase-Producing Pseudomonas aeruginosa in the United
Kingdom
Laura L. Wright1,2*, Jane F. Turton1, David M. Livermore1,2, Katie L. Hopkins1, Neil Woodford1
1Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Colindale,
Public Health England, London, NW9 5EQ, United Kingdom.
2Norwich Medical School, University of East Anglia, Norwich, Norfolk, NR4 7TJ, United
Kingdom.
Corresponding author:
Miss Laura L. Wright
Antimicrobial Resistance and Healthcare Associated Infections Reference Unit
Public Health England
61 Colindale Avenue
London NW9 5EQ
Email: [email protected]
Tel: +44(0)2083276764
Running Title: MBL-producing P. aeruginosa in the UK
Keywords: VIM, ‘high-risk clone’, ST111, ST235, carbapenemase
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SynopsisObjectives: Carbapenem-resistant isolates of Pseudomonas aeruginosa producing metallo-β-
lactamases (MBLs) are increasingly reported worldwide and often belong to particular ‘high-risk
clones’. This study aimed to characterise a comprehensive collection of MBL-producing P.
aeruginosa isolates referred to the UK national reference laboratory from UK laboratories over a 10-
year period.
Methods: Isolates were referred to the UK national reference laboratory between 2003 and 2012 for
investigation of resistance mechanisms and/or outbreaks. MBL genes were detected by PCR. Typing
was carried out by nine-locus variable number tandem repeat (VNTR) analysis and multi-locus
sequence typing (MLST).
Results: MBL-producing P. aeruginosa isolates were referred from 267 source patients and 89 UK
laboratories. The most common isolation sites were urine (24%), respiratory (18%), wounds (17%)
and blood (13%). VIM-type MBLs predominated (91% of all MBLs found) but a few IMP- and NDM-
type enzymes were also identified. Diverse VNTR types were seen, but 86% of isolates belonged to
six major complexes. MLST of representative isolates from each complex showed that they
corresponded to sequence types 111, 233, 235, 357, 654 and 773 respectively. Isolates belonging to
these complexes were received from between nine and 25 UK referring laboratories each.
Conclusions: The incidence of MBL-producing P. aeruginosa is increasing in the UK. The majority of
these isolates belong to several 'high-risk clones', which have been previously reported
internationally as host clones of MBLs.
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IntroductionPseudomonas aeruginosa is a common opportunistic pathogen responsible for many hospital-
acquired infections. Carbapenems are important antibiotics for treatment of these infections, but
resistance is increasing worldwide. Although most carbapenem resistance in P. aeruginosa arises by
mutations that lead to the loss of porin OprD or (for meropenem, not imipenem) upregulation of
efflux pumps (e.g. MexAB-OprM),1 carbapenemases are also increasingly reported. Serine
carbapenemases of the KPC, GES and OXA families have occasionally been reported in this species,
with limited geographical scatter,2–4 but metallo-β-lactamases (MBLs), particularly VIM- and IMP-
types, are predominant, and have been reported globally.5
MBLs hydrolyse almost all β-lactam antibiotics, including carbapenems, and most MBL-producing P.
aeruginosa strains are resistant to other antibiotic classes, including fluoroquinolones and
aminoglycosides, often leaving polymyxins as the sole therapeutic options. The MBL genes often
reside on mobile genetic elements able to be transmitted between strains,5 but are commonly
associated with multi-resistant ‘high-risk clones’. These have been identified in several bacterial
species and, in the case of P. aeruginosa , the most commonly reported ‘high-risk clones’ belong to
sequence types (STs) 111, 235 and 175.6 Recent studies report major dissemination of an ST235
lineage with the VIM-2 MBL in Russia, Belarus and Kazakhstan,7 and of ST277 with the SPM-1 MBL in
Brazil.8
In recent years, the number of MBL-producing P. aeruginosa referred to the UK national reference
laboratory has steadily risen. We sought to determine the contribution of internationally-recognised
‘high-risk clones’ to this increase.
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Materials and Methods
Bacterial isolatesThree-hundred and thirty-four MBL-positive isolates were identified amongst P. aeruginosa isolates
referred from UK hospital laboratories to Public Health England’s (PHE) Antimicrobial Resistance and
Healthcare Associated Infections Reference Unit (formerly the Health Protection Agency’s ARMRL
and LHCAI laboratories) between 2003 and 2012 for susceptibility testing, investigation of resistance
mechanisms, and/or strain typing. Isolates were identified as MBL-producers by PCR, either at the
time of referral (304 isolates), or by retrospective screening of isolates submitted from 2009-2012
that shared similar VNTR types with other MBL-producing isolates (30 isolates). Demographic and
isolation site data were obtained from the Laboratory Information Management System. Referring
laboratories were assigned codes to indicate the UK region and given a unique number within the
region, in the format “region_number” (e.g. North West_1).
As a comparator group, all 209 P. aeruginosa isolates collected in 2011 as part of the British Society
for Antimicrobial Chemotherapy’s (BSAC) Bacteraemia Surveillance Programme from hospitals in the
UK and Irish Republic were also studied (http://www.bsacsurv.org/).
Analysis of MBL genesGenes encoding VIM-, IMP-, SPM-, GIM-, and SIM-type MBLs were sought by multiplex PCR, as
detailed by Ellington et al.9 Genes for NDM-type MBLs were sought by a single PCR, as previously
described.10 Sequencing of blaVIM and blaIMP MBL genes was carried out using previously-described
primers specific to MBL genes and class 1 integrons;11 sequencing of blaNDM genes was with primers
NDM-orfF (5’-ATGGAATTGCCCAATATTATG-3’) and NDM-orfR (5’-TCAGCGCAGCTTGTCGGCCA-3’).
TypingPulsed-Field Gel Electrophoresis (PFGE) was the routine typing and outbreak investigation method
used in the reference laboratory for P. aeruginosa between 2003 and 2009, and was carried out
using SpeI-digested genomic DNA.12 Nine-locus variable number tandem repeat (VNTR) analysis
became the routine method from 2009 onwards, and was performed as previously described. 12
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Minimum spanning trees were produced using Bionumerics software v6.1 (Applied Maths, Sint-
Martens-Latem, Belgium). Multi-locus sequence typing (MLST) analysis was undertaken as described
by Curran et al;13 results were analysed using Bionumerics software, and sequence types (STs)
assigned using the P. aeruginosa MLST database (http://pubmlst.org/paeruginosa/).
Results
Patient demographicsThe 334 MBL-positive isolates were from a total of 267 patients, with VNTR profiles remaining
consistent when multiple isolates were received from the same patient. Accordingly, one isolate per
patient was selected for further study, leaving 267 non-duplicate-patient isolates from 89 UK
laboratories. Ages of the source patients ranged from 0-94, mean 54 yrs: proportions in age bands
were as follows: 0-2years, 4%; 2-15, 2%; 16-29, 6%; 30-44, 11%; 45-59, 22%; 60-74, 32% and >74,
12% . Predominant isolation sites were urine (24%), respiratory (18%), wounds (17%) blood (13) and
indwelling devices (7%) whereas few were from skin (3%) and faecal (3%) specimen lastly to
represent screeing rather than infection.. Sixty percent of patients were male, 35% female, and for
5% gender was not stated. blaVIM genes alone were detected in 243 isolates, blaIMP in 22, and blaNDM in
one; one further isolate had both blaVIM and blaNDM. Of the 89 referring laboratories, 83 submitted
isolates from fewer than ten patients over the ten-year period, with 71 submitting only one to two
isolates each. The remaining six laboratories, which submitted between 13 and 31 isolates each,
collectively accounted for 43% of all patients (116/267).
TypingAmong the 267 non-duplicate-patient isolates, 232 had VNTR data available, or generated in this
study, and a minimum spanning tree based on these data is shown in figure 1. The remaining 35
isolates were not available in the archive for retrospective VNTR typing, but 19 had previously-
determined PFGE profiles (not shown) identical to those of isolates with known VNTR types from a
suspected outbreak at the same hospital. These isolates were assumed to belong to the
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corresponding VNTR complex and are included in Table 1. The remaining 16/35 unavailable isolates
had no typing data, or belonged to PFGE types unique to their respective hospital; these latter
isolates, from 12 laboratories, are excluded from Table 1; all carried blaVIM.
Six VNTR complexes (designated A-F) accounted for 86% of the 251 isolates with VNTR data available
or inferred from PFGE data. Isolates belonging to these six complexes were referred from between
nine and 25 laboratories and, owing to their predominance,became the focus of further study.
Sixty-four representatives, covering the variation in VNTR profile within each complex, were selected
for MLST analysis. Isolates belonging to complexes A, B, C, D, E and F were found to belong to ST111,
ST235, ST233, ST654/ST964, ST357 and ST773, respectively (Table 1). Ten of 11 MLST-typed complex
D isolates belonged to ST654, but one belonged to ST964; this is a single locus variant (SLV) of ST654,
differing only in the acsA allele, where ST964 has allele 145 with a single C to T substitution
compared with allele 17 in ST654. Table 2 shows the distribution of each of the major complexes
among referring laboratories. The largest group of isolates was complex A (corresponding to ST111;
VNTR type 11,3,4,3,2,2,x,4,x, where x is variable), with 75 representatives. It included isolates from
25 laboratories spread across the UK, but with more than half of the isolates coming from outbreaks
at London_17 and Wales_1, referring 29 and 13 isolates, respectively, all with blaVIM. Most other
complex A isolates (28/33) also had blaVIM, but five, from two laboratories, had blaIMP. VNTR profiles
were highly conserved amongst all complex A isolates, with most only differing by repeat numbers
between six and eight at locus 61.
Complex B (ST235; 13,3,6,4,5,1,x,2,x) was the secondlargest group, comprising 52 isolates referred
from 25 different laboratories. There were potential outbreaks at sites London_13 and Scotland_2,
referring eight and six isolates, all with blaVIM; these groups were received over seven months and
two years respectively. Another laboratory (London_7) also submitted isolates carrying blaVIM from
eight patients over an eight-year period. The remaining 30 Complex B isolates were from 22
laboratories, each submitting one to three isolates; 24 isolates had blaVIM, and six had blaIMP.
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Twenty-six isolates belonged to complex C (ST233; 12,3,4,5,3,1,x,x,x). These all carried blaVIM and
were from 16 laboratories, which submitted one to four representatives each. Nineteen isolates
from 11 laboratories belonged to complex D (ST654; 11,3,2,15,3,1,x,3,x); seven of these were
submitted from South East_6 and all had blaVIM and shared an identical VNTR profile, whilst one or
two isolates were referred from each of the remaining ten laboratories; ten isolates had blaVIM, one
had blaIMP, and one blaNDM. Most of the 30 complex E isolates (ST357; 13,2,1,5,2,3,6,5,x), all with
blaVIM, were from an outbreak at North West_15, with 22 isolates referred over a nine-year period;
12 of these 22 were submitted in 2007. Each of the remaining eight complex E isolates was referred
from a different laboratory. Finally, 13 isolates, all with blaVIM, belonging to complex F (ST773;
12,4,6,5,3,1,10,x,x) were received from 11 laboratories in diverse areas of the UK.
The diversity of carbamenemes gene sequences was investigated by testing representatives of the
VNTR variationwithin the six major complexes. Irrespective of VNTR variation all blaVIM-–positive
representatives of complexes A, C, D, E and F (STs 111, 233, 654, 357 and 773) had blaVIM-2, whereas
the eight isolates selected from complex B (ST235) variously had blaVIM-1 (2 isolates), blaVIM-2 (4),
blaVIM-4 (1) and blaVIM-6 (1). blaIMP-1 (3 isolates), blaIMP-7 (1), blaIMP-10 (1) and blaIMP-13 (2) alleles were seen
amongst seven representatives of the 22 blaIMP positive isolates variously belonging to complexes
XXXXXXXXXXXX,. Both blaNDM positive isolates, one belonging to complex D, the other a ‘diverse’
strain, had the blaNDM-1 allele.
The 14% of isolates (n=36) that did not belong to any of these six major complexes were diverse by
VNTR, with 26 different VNTR profiles represented. They were referred from 24 laboratories across
the UK, each submitting one to three isolates each; 26 had blaVIM, 10 had blaIMP and one had both
blaVIM and blaNDM genes.
Regional distribution of MBL-positive isolates The UK distribution of the 267 non-duplicate-patient isolates is shown in figure 2. London accounted
for 47% of these organisms, with MBL-positive isolates referred from 28 London-region laboratories;
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all six major complexes (A-F) were represented and nine other VNTR types seen. Four London
laboratories were amongst the six sites that submitted more than ten MBL-positive isolates.
London_17 submitted 29 complex A (ST111) isolates over six years, with source patients on multiple
wards. London_13 had separate outbreaks of MBL-positive isolates belonging to complexes B and C
(STs 235 and 233), with eight and four representatives each, respectively. Each “outbreak” lasted
one to two years separated by a six-year gap. London_7 and London_12, referred 19 and 15 isolates,
respectively, over seven to eight years, with isolates belonging to diverse VNTR complexes.
Greater Manchester accounted for 12% of isolates (31/267). Isolates belonging to complex E (ST357)
predominated and included all 22 from an outbreak at North West_13, although isolates belonging
to complexes A, B and D (STs 111, 235 and 654) also were seen. In Sussex, Surrey and Kent half of
the 18 isolates received belonged to complex D (ST654); seven of these 18 were from South East_6;
the remaining nine belonged to complexes B and C (STs 235 and 233) or had other VNTR profiles.
Wales accounted for 6% of isolates, mostly (13/15) from an outbreak of ST111 P. aeruginosa at
Wales_1 although one ST111 isolate was from another laboratory, and one ST773 isolate also was
referred from the region. Finally, the 12 (4%) isolates submitted from Scotland belonged to
complexes A, B, C and F (STs 111, 235, 233 and 773). Fewer than ten isolates were referred from
each of the remaining UK regions.
Typing of comparator P. aeruginosa isolatesThe comparator set, comprising P. aeruginosa isolates collected as part of the BSAC Bacteraemia
Resistance Surveillance Programme in 2011 showed far greater diversity than the MBL producers,
with 136 different VNTR profiles represented amongst the 209 isolates (figure 3). Although 4·8% (10
isolates) were imipenem resistant, none produced MBLs. Two isolates shared VNTR profiles with
complex B (ST235) and one shared a profile with complex C (ST233); the remaining 206 isolates did
not share VNTR profiles with any of the major complexes identified amongst the MBL-producing
isolates.
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Discussion
Most (86%) MBL-producing P. aeruginosa isolates referred to the UK reference laboratory between
2003 and 2012 belonged to six VNTR complexes, corresponding to internationally recognised ‘high-
risk clones’, STs 111, 235, 233, 357, 654 and 773, and most had VIM-type MBLs. These findings are in
striking contrast to the general population structure of P. aeruginosa, where a recent UK study14
shows considerable diversity, with overlap between environmental isolates and those causing
clinical infection, albeit with a few prevalent clusters in diverse locations. The major complexes seen
here were not prevalent amongst MBL-negative isolates in this previous study, nor were they
prevalent in our comparator collection of (largely susceptible) P. aeruginosa isolates from the BSAC
Bacteraemia Resistance Surveillance Programme. Rather, these ‘high-risk clones’ seem to represent
a distinct subset of P. aeruginosa lineages, which may be successful precisely due to a particularly
strong ability to acquire and/or maintain resistance genes compared with the general P. aeruginosa
population. Notably, the reference laboratory has also received MBL-negative representatives
corresponding to these six major complexes, including ST235 isolates carrying blaGES-5 and a ST773
isolate with blaOXA-181, both genes encoding non-metallo-carbapenemases (unpublished data; J.
Turton, K. Hopkins). Moreover, in three of these complexes, we encountered isolates that variously
had either blaVIM or blaIMP genes, and in one complex (XXX), various different blaVIM alleles.
Each of the six main VNTR complexes has been reported internationally as a host for MBLs. Complex
A (corresponding to ST111) was the largest group in our collection, with most isolates carrying blaVIM-
2. VNTR profiles within this complex were highly similar; most differing only at locus 61, which is the
most variable of the nine VNTR loci.12 ST111 is frequently recorded amongst MBL-producing P.
aeruginosa, with ST111 isolates reported to be of serotype O12, which has been recognised as a
common host of multiresistance in Europe since the 1980s.15,16 ST111 was the predominant ST type
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seen in a nationwide study of MBL-producing P. aeruginosa in The Netherlands in 2010-11,16 and
was responsible for outbreaks affecting hospitals in central Greece,17 and Italy.18 In all these settings,
as in the UK, the ST111 isolates generally carried blaVIM-2. Smaller numbers of ST111 isolates with
blaVIM-2 have been reported in other European countries including Sweden,11 Croatia,19, Spain20 and
Germany,21 whilst isolates with this ST have been associated with blaVIM-4 in Hungary,22 blaVIM-1 in
Greece23 and blaIMP-13 in France.24 Outside Europe, an isolate of ST111 from a patient in Colombia was
recently found to carry both blaVIM-2 and blaKPC-2.25
The second-most-prevalent complex (B) in our collection corresponded to ST235. Isolates in this
complex had more diverse VNTR profiles than those in complex A, and were more widely
disseminated among referring laboratories, with no more than eight from any single site. Again,
most had blaVIM, but with diverse blaVIM alleles identified. Together with the VNTR diversity, this
carbapenemase diversity suggests multiple imports and/or acquisitions of blaVIM by ST235 P.
aeruginosa. This variation is in contrast to the ST235 clone with blaVIM-2 that is widespread across
Russia, Belarus and Kazakhstan.7 ST235 was the most commonly identified MBL-producing P.
aeruginosa type in a study of five Mediterranean countries,26 and ST235 isolates with blaVIM-2 genes
have also been reported in Spain,20 Croatia,19 Germany,21 and Greece,23 blaVIM-4 in Hungary,22
Norway,11 and Belgium,27 and blaVIM-13 in Spain.28 In Asia, outbreaks of ST235 clones with blaIMP-6 genes
have occurred in Japan,29 and South Korea,30 with blaVIM-positive isolates with this ST also seen in
Thailand, Malaysia, Sri Lanka and Korea.31 There are single reports of ST235 isolates carrying blaNDM-1
and blaSPM-1 genes in France,32 and Brazil,8 respectively.
The other four major complexes (C-F) also correspond to ‘high-risk clones,’ previously reported as
hosts for MBLs. ST233 (complex C) isolates with blaVIM-2 have been found in Norway (imported from
Ghana),11 Japan,33 South Africa,34 and carrying a blaIMP-1 variant in Singapore.35 We likewise
consistently saw blaVIM-2 in complex C isolates. ST654 (complex D) has been reported in Sweden
(imported from Tunisia) carrying blaVIM-2 genes,11 in Singapore carrying blaIMP-1 and blaIMP-26,35 and in
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Argentina carrying a KPC carbapenemase.4 We predominantly saw blaVIM-2 in ST654 isolates, but one
isolate had blaNDM-1. The sole ST964 isolate (also complex D) had blaIMP-1, as also reported in this ST in
Singapore.35 We saw only blaVIM-2 in representatives of complex E (ST357), but this ST has previously
been reported with blaVIM-2 or blaIMP-7 genes in a hospital in the Czech Republic,36 whilst a few ST357
isolates with blaIMP-7 were reported in Poland.37 ST773 (complex F) has been recently reported among
Indian isolates carrying blaVIM-2.31 Interestingly, complex F isolates tested here also all had blaVIM-2 and
four isolates were from patients who had recently travelled to India; travel history for the other nine
patients was not available. The remaining VNTR profiles, accounting for 14% of MBL producers, were
diverse and probably represent separate acquisitions of blaVIM and blaIMP genes.
Ongoing research indicates a variety of VIM-containing class 1 integron structures amongst the MBL
producers. Complexes A, D, E and F (STs 111, 654, 773 and 357) typically have a single predominant
blaVIM-2 carrying integron, whereas a different (but converved) blaVIM-2-carrying integron was seen in
all complex C isolates and some belonging to complex B (STs 233 and 235) (Wright et al.,
unpublished data).
Although comprehensive epidemiological data are lacking, VIM-type MBLs generally are the
predominant carbapenemases seen in P. aeruginosa in Europe, with only sporadic isolations, and/or
local spread of strains producing IMP- or NDM- types. The 22 isolates harbouring blaIMP genes
belonged to diverse VNTR types, with diverse blaIMP alleles and no known epidemiological links
between most isolates. Just two blaNDM-1-positive isolates were found, both with different VNTR
types. These IMP- and NDM-MBL-producing isolates may have been imported from outside the UK,
or acquired locally, with local spread at a small number of sites. Unfortunately, data on patient travel
were not available for most isolates, but single isolates carrying blaNDM and blaIMP were from patients
who had travelled to India and Pakistan, respectively.
MBL-positive P. aeruginosa were referred from around half the hospital laboratories in the UK, with
all six major complexes found in multiple UK regions. Referral of suspect isolates is not mandatory,
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and some likely MBL producers were no longer viable in our archives, so the numbers reported here
under-estimate the true incidence of MBL-producing P. aeruginosa. They were rare at most referring
sites, but a few sites did have persistent problems with single clones. These include an outbreak of
ST111 isolates at London_17 and of ST654 isolates at South East_6, both associated with
contamination of the waste-water networks,38 and an outbreak of ST357 P. aeruginosa at North
West_15 where the strain, which also produced a VEB-1a extended-spectrum-β-lactamase, was
thought to have been imported via a patient transferred from an Indian hospital, but to have
acquired the VIM-MBL locally in the UK.39 In contrast, two laboratories (London_7 and London_12)
referred MBL-positive isolates of diverse types over seven and eight year periods, implying that MBL-
producing P. aeruginosa had been introduced repeatedly.
Since these ‘high-risk clones’ are reported amongst MBL-producing P. aeruginosa worldwide, it is
important not to assume that UK isolates with the same VNTR or ST profile are directly related
unless this view is supported by evidence of epidemiological links between affected patients; it is
just as likely that cases could represent repeated imports of the same clone from different sources.
Although, MBL-producing P. aeruginosa have so far caused outbreaks at few UK hospitals, those that
have occurred have been linked to environmental reservoirs within the hospitals, including waste-
water networks, as well as to patient-to-patient cross-infection.
AcknowledgmentsWe thank staff at the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit
for carrying out PFGE and some of the VNTR typing and MBL detection, performed as part of the
reference service. We are grateful to the UK hospital laboratories for submitting isolates to us and to
the British Society for Antimicrobial Chemotherapy for allowing us to access isolates from their
Bacteraemia Surveillance Programme. Part of this work was presented at the 53rd ICAAC, 2013,
Denver, USA (Abstract: C2-1597).
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FundingThis work was supported by Public Heath England through a competitively awarded PhD
studentship.
Transparency DeclarationsN.W. and D.L. have received research grants and/or fees from various pharmaceutical companies.
D.L. also holds shares in several pharmaceutical companies. None of these poses a conflict of
interest with this work. All other authors; none to declare.
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Table 1: Typing data for the six main VNTR complexes identified (n=251)
VNTR Complex
VNTR type a No. of different VNTR profiles
MLST type(s) (no. of isolates tested)
No. of isolatesb
No. of submitting laboratories
MBLs detected (no. of isolates)
A 11,3,4,3,2,2,x,4,x 6 ST111 (11) 75 25 VIM (70)
IMP (5)
B 13,3,6,4,5,1,x,2,x 16 ST235 (18) 52 25 VIM (46)
IMP (6)
C 12,3,4,5,3,1,x,2,x 11 ST233 (10) 26 16 VIM (26)
D 11,3,2,15,3,1,x,3,x 6 ST654 (10), ST964 (1)
19 11 VIM (17)
IMP (1)
NDM (1)
E 13,2,1,5,2,3,6,x,x 7 ST357 (9) 30 9 VIM (30)
F 12,4,6,5,3,1,10,x,x 3 ST773 (5) 13 11 VIM (13)
Others Diverse 26 Not done 36 25 VIM (25)
IMP (10)
VIM and NDM (1)
ax represents loci where the repeat number varies between isolates within a complex
bone isolate per patient was included; these numbers include four isolates (complex B), 14 isolates
(complex E) and one isolate (complex F) where the MBL-positive organisms were no longer available
in the archive for VNTR analysis, but which were previously found to share a PFGE profile, and are
from the same hospital outbreak as other isolates in the respective complex. Isolates were also
received from an additional 39 patients at London_17 with a PFGE profile corresponding to complex
A. These are not included here as they had not been screened for MBL genes and were no longer
available in our archives.
396
397
398
399
400
401
402
403
404
405
Table 2: Geographic and temporal distribution of MBL-producing P. aeruginosa belonging to the six
major complexes (215 isolates) among referring laboratories
VNTR Complex
Major Contributors (≥ 5 referred isolates) Minor Contributors
Referring laboratorya
Number of isolatesb
Time period over which isolates referred
MBL genes detected
A (n=75) London_17 c 29 (39%) 80 months all with blaVIM 2 laboratories with 4 representatives each
Wales_1 13 (17%) 18 months all with blaVIM 2 laboratories with 3 representatives each
19 laboratories with 1 representative each
B (n=52) London_7 8 (15%) 87 months all with blaVIM 1 laboratory with 3 representatives each
London_13 8 (15%) 7 months all with blaVIM 6 laboratories with 2 representatives each
Scotland_2 6 (12%) 27 months all with blaVIM 15 laboratories with 1 representative each
C (n=26) London_7 5 (19%) 27 months all with blaVIM 3 laboratories with 2 representatives each
London_13 4 (15%) 18 months all with blaVIM 11 laboratories with 1 representative each
D (n=19) South East_6 7 (37%) 36 months all with blaVIM 2 laboratories with 2 representatives each
8 laboratories with 1 representative each
E (n=30) North West_15 22 (73%) 103 months all with blaVIM 8 laboratories with 1 representative each
F (n=13) No major contributors 2 laboratories with 2 representatives each
9 laboratories with 1 representative each
areferring laboratories are coded in the format ‘UK region_number’.
bone isolate per patient is included; these numbers include four isolates (complex B), 14 isolates
(complex E) and one isolate (complex F) that were not available in the archive for VNTR analysis, but
406
407
408
409
410
which shared a PFGE profile and are from the same hospital outbreak as other isolates in the
complex.
c Isolates were also received from an additional 39 patients at London_17 with a PFGE type
corresponding to complex A. These are not included here as they had not been screened for MBL
production and were no longer available in our archives.
Figure 1: Minimum spanning tree, based on clustering at the first eight VNTR loci for MBL-positive P.
aeruginosa, with one isolate included per patient (n=232). The six main complexes A-F are labelled.
Coloured segments of the circles indicate laboratories that submitted three or more isolates, white
segments represent laboratories submitting one or two isolates. The diameter of the circle is relative
to the number of isolates with that VNTR profile. Shading indicates complexes. Thick solid lines
represent single locus variants; thin solid lines and dotted lines represent multi-locus variants.
Figure 2: Geographical sources of isolates in the UK, and distribution of the six main complexes (A-F)
in each of the five regions referring more than ten isolates. This figure appears in colour in the online
version of JAC and in black and white in the print version of JAC.
Figure 3: Minimum spanning tree based on clustering at the first eight VNTR loci for P. aeruginosa
isolates from the BSAC Bacteraemia Resistance Surveillance Programme (n=209). The diameter of
the circle is relative to the number of isolates with that VNTR profile. Shading indicates complexes.
Thick solid lines represent single locus variants while thin solid lines and dotted lines represent multi-
locus variants. Isolates corresponding to VNTR complexes B and C (STs 235 and 233) are shown in
black circles. Isolates corresponding to previously reported clones as described by Martin et al.14 are
indicated and had the following VNTR profiles; Clusters A (8,3,4,5,2,3,5,2,x), D (10,3,5,5,4,1,3,x,x,), E
(11,4,5,2,2,1,x,2,x) and H (12,5,1,5,2,2,x,x,x), Clone C (11,4,5,2,2,1,x,2,x), and PA14
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
(12,2,1,5,5,2,x,5,x). Two novel clusters are indicated with VNTR profiles (12,5,5,5,4,3,7,6,x) and
(12,8,2,2,4,3,5,1,x) respectively.
435
436
437
Figure 2438
439
Figure 3
1
2
2
2
3
4
2
3
6
3
3
7
5
5
2
5
6
28
1
2
2
2
3
5
5
6
7
7
7
9
9
12
15
18
31
126
0 50 100 150
Devon, Cornwall and Somerset
Avon, Gloucestershire and Wiltshire
North East
Northern Ireland
Bedfordshire, Hertfordshire andNorthamptonshire
Hampshire, Isle of Wight and Dorset
Lincolnshire, Leicestershire,Nottinghamshire and Derbyshire
Thames Valley
Cheshire and Merseyside
Cumbria and Lancashire
Norfolk, Suffolk, Cambridgeshire and Essex
West Midlands
Yorkshire and Humber
Scotland
Wales
Sussex, Surrey and Kent
Greater Manchester
London
Isolates
Referring hospitals
440
441
Figure 4
PA14 (ST253)
Cluster H
Clone C (ST17)
Complex B (ST235)
Complex C (ST233)
Cluster A (ST27) Cluster D
(ST395)Cluster E
Novel cluster 1
Novel cluster 2
442
443