RESEARCH ARTICLE

Identi¢cation of PER-1extended-spectrum b-lactamase producing

Pseudomonas aeruginosa clinical isolates ofthe international clonal
complex CC11from Hungary and Serbia
Balázs Libisch1, Laurent Poirel2, Zorica Lepsanovic3, Veljko Mirovic3, Boglárka Balogh1, Judit Pászti4,
Zsuzsanna Hunyadi4, András Dobák5, Miklós Füzi1 & Patrice Nordmann2
1
Department of Bacteriology, National Center for Epidemiology, Budapest, Hungary; 2Service de Bactériologie-Virologie, Hôpital de Bicêtre, Faculté de
Médecine Paris-Sud, Université Paris Sud, France; 3Department of Epidemiology, Military Medical Academy, Belgrade, Serbia; 4Department of Molecular
Epidemiology, National Center for Epidemiology, Budapest, Hungary; and 5Prodia Microbiological Laboratory, Budapest, Hungary

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Correspondence: Balázs Libisch,
Department of Bacteriology, National Center
for Epidemiology, H-1097 Budapest, Gyáli u
2-6, Hungary. Tel.: 136 1 476 1118;
fax: 136 1 476 1234; e-mail:
balazs.libisch@freemail.hu
Received 15 April 2008; revised 5 August 2008;
accepted 29 August 2008.
First published online 16 October 2008.
DOI:10.1111/j.1574-695X.2008.00483.x
Editor: Kai Man Kam
Keywords
PER-1; Pseudomonas aeruginosa ; OXA-74;
fluroquinolone acetyltransferase; CC11; MLST.
Abstract
PER-1 extended-spectrum b-lactamase-producing Pseudomonas aeruginosa clin-
ical isolates from Budapest, Hungary, and Belgrade, Serbia, were characterized by
molecular methods. Two PER-1-positive isolates were recovered from sporadic
cases in Budapest and a small cluster of PER-1-positive infections involving four
patients were identified at a Belgrade hospital. A class 1 integron harbouring a
blaOXA-2 b-lactamase gene and four other gene cassettes was detected in both the
Budapest and the Belgrade isolates. The two P. aeruginosa isolates from Budapest
also carried another class 1 integron containing blaOXA-74, aac(6 0 )-Ib-cr and cmlA7
genes in its variable region. The aac(6 0 )-Ib-cr fluoroquinolone-acetylating amino-
glycoside acetyltransferase gene is described here for the first time in P. aeruginosa.
Multilocus sequence typing (MLST) revealed that the PER-1 positive P. aeruginosa
isolates identified in this study display ST235, a sequence type that belongs to
clonal complex CC11. Two blaPER-1-positive P. aeruginosa reference isolates from
France and Belgium could also be assigned to complex CC11 by MLST. Our results
underscore the role of complex CC11 in the dissemination of blaPER-1 among
P. aeruginosa clinical isolates.

Turkish patient and was found to be widespread among

Introduction
Pseudomonas aeruginosa is commonly responsible for no-
socomial infections, including surgical site infections,
urinary tract infections, pneumonia and bloodstream infec-
tions. Acquired b-lactamases have frequently been reported
from this species, with the IMP- and VIM-type metallo
b-lactamases, the PER, VEB and GES-type extended-spectrum
b-lactamases (ESBLs) as some of the major types (Weldhagen
et al., 2003; Endimiani et al., 2006). ESBLs in P. aeruginosa
usually confer resistance to all b-lactams but not to carbape-
nems (with the exception of certain enzymes such as GES-2)
and the proportion of ESBL-producing isolates is increasing
globally (Paterson & Bonomo, 2005; Nordmann et al.,
2007). The PER-1 ESBL is an Ambler class A b-lactamase
conferring a high level of resistance to the broad-spectrum
b-lactams and is one of the most frequently detected ESBLs
in P. aeruginosa. PER-1 was first identified in France from a
Acinetobacter spp. and P. aeruginosa isolates in Turkey. Since
then, PER-1 has been discovered in several other countries,
such as Belgium, Italy and Spain and recently has also been
found in Korea, Romania and Bulgaria (Nordmann et al.,
1993; Yong et al., 2003; Naas et al., 2007; Strateva et al.,
2007).
Recently, 41 isolates producing the PER-1 enzyme were
recovered from a hospital in Warsaw, Poland, that clustered
to three separate clones (Empel et al., 2007). Two of these
clones corresponded to sequence types (STs) ST244 and
ST235 by multilocus sequence typing (MLST), and were
responsible for parallel outbreaks. Apart from PER-1, all the
isolates produced an OXA-2 type oxacillinase and the ST235
isolates additionally expressed a novel b-lactamase, OXA-74.
According to eBURST analysis of the currently available
STs in the P. aeruginosa MLST database (http://pubmlst.org/
paeruginosa/), ST235 is the ancestral or the founder strain

c 2008 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 54 (2008) 330–338
Published by Blackwell Publishing Ltd. All rights reserved
PER-1-producing P. aeruginosa from Hungary and Serbia 331

type of an international clonal complex initially described as
BG11 (Giske et al., 2006). This clonal complex was demon-
strated to be involved in the dissemination of blaVIM and
blaPER-1 in certain European hospitals and already contains
mainly serotype O11 P. aeruginosa isolates from Italy,
Greece, Sweden, Hungary, Poland, Russia, Turkey and the
United States (Giske et al., 2006; Empel et al., 2007; Libisch
et al., 2008).
A collection of ceftazidime-resistant P. aeruginosa clinical
isolates representing all regions of Hungary and also the
Military Medical Academy (MMA) hospital in Belgrade,
Serbia, were screened for the production of ESBL enzymes
system (BioMérieux, France) and by Micronaut-E (Gen-
zyme Virotech, Rüsselsheim, Germany).
Antibacterial susceptibility tests and detection
of ESBLs by phenotypic methods
Test organisms were inoculated onto plates of Mueller–
Hinton agar (Oxoid, Basingstoke, UK) as recommended by
the Clinical and Laboratory Standards Institute (CLSI,
2008). Minimal inhibitory concentrations (MICs) were
determined by the agar dilution method (CLSI, 2008) for
b-lactam antibiotics and by the Etest (AB Biodisk, Solna,

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by phenotypic and molecular methods. The aim of our
study was to identify the ESBL determinants among these
P. aeruginosa clinical isolates from Hungary and Serbia and
to determine their clonal relationship with the recently
described international P. aeruginosa clonal complexes.
Sweden) for other antibiotics. Antimicrobial susceptibility
test discs were purchased from Oxoid (Basingstoke, UK). To
detect ESBL production, a disk synergy test was used as
described previously (Pagani et al., 2004) with the addition
of a 75 mg cefoperazone disk.

Materials and methods
Bacterial strains
Altogether, 144 ceftazidime nonsusceptible P. aeruginosa
clinical isolates representing all geographical regions of
Hungary obtained between January 2002 and October 2007
were screened for ESBL production, together with 31
ceftazidime nonsusceptible isolates from the MMA hospital
in Belgrade, Serbia. The rates of nonsusceptibilty for cefta-
zidime among P. aeruginosa clinical isolates in Hungary and
the MMA hospital in 2006 were about 11.2% and 37%,
respectively (http://www.oek.hu; Z. Lepsanovic, unpub-
lished data). The blaPER-1-positive P. aeruginosa isolates
characterized in this study are listed in Table 1, together
with their features. The P. aeruginosa strains Pa695, 10.2 and
RNL-1 were used as reference GES-1-, VEB-1- and PER-1-
producing strains, respectively (Nordmann et al., 1993;
Dubois et al., 2002; Aubert et al., 2004). The ATCC 27853
P. aeruginosa strain was used as the quality control strain.
Identification of isolates was carried out by the API 20 NE
Molecular techniques
Detection of blaPER genes by PCR and their sequencing
were performed using the primers listed in Table 2. PER-
UPST and PER-DOWN primers recognize sequences that
are flanking the blaPER-1-coding region and were designed
against nucleotides 1346–1366 and 2386–2408 of the
P. aeruginosa transposon Tn1213 with accession no.
AY779042 (Poirel et al., 2005). The oligonucleotides used
for amplifying and sequencing of integrons were those
described in Table 2 and by earlier works (Libisch et al.,
2006).
Mating-out assay and plasmid analysis
Mating-out assays were carried out on Mueller-Hinton
(MH) agar plates and transconjugants were selected on agar
containing 16 mg mL1 ceftazidime and 300 mg mL1 rifam-
picin using the RifR Pseudomonas putida strain UWC1 as a
recipient (Gotz & Smalla, 1997). The initial donor/recipient
ratio was 0.3. Mating plates were incubated at 37 1C for 14 h.
Conjugation was also attempted in Luria–Bertani broth at

Table 1. Various features of the PER-1-producing Pseudomonas aeruginosa isolates from Hungary and Serbia
Date of Patient’s Age PFGE Integron
Strain City Hospital isolationw Ward sex (years) Sample Serotype type contentz
05-176 Belgrade MMA 08/2004 Dermatology M 47 Wound swab O11 A1 B, C
05-179 Belgrade MMA 10/2004 Plastic surgery M NA Wound swab O11 A2 B, C
05-181 Belgrade MMA 01/2005 Plastic surgery F 46 Wound swab O11 A3 B, C
05-279 Belgrade MMA 06/2005 Dermatology F 66 Ulcer swab O11 A4 B, C
06-116 Budapest OITI 07/2002 ICU M 40 Nasal swab O11 B2 A, B
05-380 Budapest OBSI 09/2005 ICU F 23 Trachea O11 B1 A, B
Hospitals are indicated with the abbreviation of their Hungarian name.
w
Date of isolation is given as mm/yyyy.
z
Integrons A, B and C correspond to those in Fig. 1 with accession nos, EU161636, EU165039 and EU165040, respectively.
NA, not available.

FEMS Immunol Med Microbiol 54 (2008) 330–338

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
332 B. Libisch et al.

37 1C for 14 h with the same donor/recipient ratio. Plasmid PFGE and serotyping
analysis was performed by the method developed by Kado
PFGE was performed according to the method described by
& Liu (1981), using the PureLinkTM HiPure Plasmid DNA
Poh with modifications (Poh et al., 1992). Genomic DNA
Purification Kit (Invitrogen) and also by pulsed-field gel
inserts were digested at 37 1C for 2.5 h with 20 U of SpeI
electrophoresis (PFGE) of undigested whole-DNA prepara-
enzyme. Electrophoresis was performed in a CHEF-DRII
tions, as described previously (Delissalde & Amábile-
apparatus (Bio-Rad, Richmond, CA). DNA fingerprints were
Cuevas, 2004). Horizontal transfer of blaPER-1 genes and
compared with the FINGERPRINTING II INFORMATIXTM software
integrons was examined by PCR analysis of the transconju-
(Bio-Rad) using a 1% band position tolerance and a cut-off
gants obtained.
value of 80% for genetic relatedness by the Dice coefficient to
identify PFGE genotypes (Giske et al., 2006). The anti-P.
aeruginosa in vitro agglutinating sera (Bio-Rad, Marnes-la-
Plasmid curing assays

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Coquette, France) were used for the detection of serotypes.
Plasmid curing was performed by incubating isolates 05-380
and 06-116 for 2 weeks at 4 1C suspended in water (Jackson MLST
et al., 1999). Phenotypic screening for cured colonies was
MLST was performed according to the protocol published
carried out by the ability of isolated colonies to grow on MH
by Curran et al. (2004). Nucleotide sequences were deter-
agar plates containing 128 mg mL1 ceftazidime. Isolates not
mined for internal fragments of the acsA, aroE, guaA, mutL,
growing on this screen agar were subjected to serotyping,
nouD, ppsA and trpE genes on both strands and searched
plasmid profile analysis and PCR mapping to detect the
against the MLST database (http//:pubmlst.org/paeruginosa/)
presence or absence of the blaPER determinant and class 1
for assignment of allelic numbers and STs. The eBURST
integrons. Also, cured and plasmid-containing isolates were
software was used for phylogenetic analysis as described
compared with random amplification polymorphic DNA
(Feil et al., 2004). Clonal complexes were defined as a group
(RAPD) typing as described (Giske et al., 2006).
of isolates with either identical STs or STs that varied
at one or two loci (single- or double-locus variants) (Curran
Table 2. List of oligonucleotide primers used for the PCR amplification et al., 2004). The clonal complex CC11 discussed in this
and sequencing of blaPER determinants and class 1 integrons
work corresponds to the previously described BG11 com-
Target Designation Primer sequence plex (Giske et al., 2006; Empel et al., 2007; Libisch et al.,
blaPER PER-F 5 0 -GCCTGACGATCTGGAACCTT-3 0 2008).
PER-R 5 0 -GCCGTCCATCAGGCAACA-3 0 The nucleotide sequences of the variable region of class 1
Tn1213 PER-UPST 5 0 -TTAGATCACGAATGAAGCACC-3 0 integrons A, B and C (Fig. 1) were deposited in GenBank
PER-DOWN 5 0 -AACCTACTCCAATTTCAAACCAT-3 0 under accession nos EU161636, EU165039 and EU165040,
aacA4 aacA4-F 5 0 -GACCTTGCGATGCTCTATG-3 0
respectively.
aacA4-R 5 0 -CAGCAACTCAACCAGAGC-3 0
aadB aadB-F 5 0 -AAGCACGATGATATTGATCTGA-3 0
aadB-R 5 0 -TATGTGCTTTGTAGGCCAGT-3 0 In vitro phenotypic assays for the synergistic
aadA2 AADA2-R 5 0 -AAGGGTGACTTCTATAGCG-3 0 activity of phenylalanine arginine
aphA15 aphA15-F 5 0 -GTCTCGACTTCAACTGTCA-3 0 b-naphthylamide (PAbN) combined with
aphA15-R 5 0 -ATGAAGCCGACGAAGGCA-3 0 levofloxacin and ciprofloxacin
blaOXA  10 OXA10like-F 5 0 -GGTGTCATAAAGAATGAGCAT-3 0
OXA10like-R 5 0 -TCCATGTTAAAGGCGAAAAAGT-3 0 The agar dilution method was used to determine MICs for
blaOXA  2 OXA2-F 5 0 -ATGGCAATC CGAATCTTC G-3 0 ciprofloxacin and levofloxacin plus/minus the Mex efflux
OXA2-R 5 0 -TTGACCAAGCGCTGATGTT-3 0 pump inhibitor PAbN, also known as MC-207,110
cmlA7 CHL-R 5 0 -CGGGTT TCAGGCCAGAA-3 0
(Lomovskaya et al., 2001), at the concentration of 50 mg mL1
OrfD ORFD-R 5 0 -CAGCGTTTGAGAAACTGAATG-3 0
(Mesaros et al., 2007).

aac(6 ′)-
(A) 5′-CS blaOXA-74 Ib-cr cmlA7 3′-CS Fig. 1. Schematic diagrams of the variable
region of class 1 integrons. (A) Strain 05-380,
Hungary. Accession no. EU161636. (B) Strains
(B) 5′-CS aadB aphA unknown blaOXA-2 orfD 3′-CS 05-380 and 05-279, Hungary and Serbia,
respectively. Accession no. EU165039. (C) Strain
05-279, Serbia. Accession no. EU165040. Empty
ellipses represent the attI1 site, black circles the
(C) 5′-CS aacA4 aadA2 3′-CS 59-base elements. 5 0 -CS and 3 0 -CS stand for the
5 0 and 3 0 conserved sequences, respectively.

c 2008 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 54 (2008) 330–338
Published by Blackwell Publishing Ltd. All rights reserved
PER-1-producing P. aeruginosa from Hungary and Serbia 333

Phenotypic detection of AmpC-mediated Characterization of class 1 integrons from the

resistance
The agar dilution method was performed using phenylboro-
nic acid as an AmpC inhibitor at the concentration of
200 mg mL1 alone and in combination with ceftazidime to
detect AmpC-mediated ceftazidime resistance, as recom-
mended (Beesley et al., 1983; Hope et al., 2008). With this
method a Z8-fold synergy (MIC potentiation ratio) with
the boronic acid/ceftazidime combination was reported for
AmpC-producing Pseudomonas spp. isolates (Hope et al.,
2008).
PER-1-producing isolates
PCR mapping experiments revealed that the two PER-1-
positive isolates from Budapest carried two class 1 integrons
of about 3.2 kb. All four isolates from Belgrade possessed a
class 1 integron of about 3.2 kb and another one of about
1.5 kb (Table 1; Fig. 1). One of the 3.2-kb integrons from
isolate 05-380 has the blaOXA-74 gene in the first position,
followed by an aac(6 0 )-Ib-cr cassette. This latter gene
encodes a fluoroquinolone-acetylating aminoglycoside acet-
yltransferase displaying Trp102Arg and Asp179Tyr substitu-

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Results
Detection of ESBLs by phenotypic methods
A collection of P. aeruginosa clinical isolates nonsusceptible
to ceftazidime and provided by Hungarian regional clini-
cal microbiology laboratories and the MMA hospital in
Belgrade between July 2002 and October 2007 were screened
for ESBL production. Table 1 shows various features of the
six ESBL-positive isolates that were identified in this study.
The ESBL disk synergy test was positive for all isolates listed
in Table 1.
Antibiotic susceptibility
MICs for most of the anti-Pseudomonas drugs are listed
in Table 3. Three of the Belgrade isolates were sensitive to
piperacillin–tazobactam according to the current CLSI
breakpoints. It is notable that only one isolate (05-176) was
sensitive to polymyxin B.
Detection by PCR and sequencing of bla PER-type
ESBL genes
PCR and sequencing experiments demonstrated that all
isolates positive by the ESBL phenotypic tests were blaPER-1
positive. The blaPER-1 allele was identified in a Tn1213-like
transposon as described previously (Mantengoli & Rossolini,
2005; Poirel et al., 2005).
tions compared with aac(6 0 )-Ib (Robicsek et al., 2006a, b).
The third cassette corresponded to a cmlA7 gene encoding a
chloramphenicol acetyltransferase (Riccio et al., 2003).
The second 3.2-kb integron from isolate 05-380 was also
present in the Belgrade isolates (Fig. 1b). This class 1
integron carries an aadB and an aphA gene in the first two
positions, where the latter gene encodes an AphA15-like
enzyme with three amino acid substitutions in its deduced
protein sequence compared with AphA15 (Riccio et al.,
2002). The third cassette contained a putative ORF of 291
nucleotides encoding a 97-amino-acid-long hypothetical
protein with no significant homology to any proteins of
known function in the GenBank database according to a
BLAST search at the time of submission of the manuscript.
This putative ORF is followed by a blaOXA-2 and an orfD
cassette.
Besides this common integron shared between the Bel-
grade and Budapest isolates, a 1.5-kb integron carrying
aacA4 and aadA2 aminoglycoside resistance genes (Fig. 1c)
was present in all four PER-1-producing isolates from the
MMA hospital in Serbia (Table 1).
Mating out assay, plasmid content analysis and
plasmid curing
For isolates 05-380 and 06-116 from Budapest, the blaPER-1
gene proved to be transferable into the P. putida recipient
strain UWC1 by in vitro conjugation experiments while
transconjugants were not obtained with the Belgrade isolates

Table 3. Antibiotic MICs for PER-1-producing Pseudomonas aeruginosa isolates from Hungary and Serbia
MIC (mg mL1)

Isolate IPM MEM ATM CAZ CAZ/CLA FEP TZP GEN AMK TOB CIP LEV CHL PO
05-176 16 4 32 4 256 4 256 16 32 128 4 256 64 4 256 4 16 4 256 2
05-179 8 4 32 4 256 4 256 16 32 16 4 256 8 4 256 4 16 4 256 4
05-181 16 4 32 4 256 4 256 16 128 64 4 256 32 4 256 4 16 4 256 8
05-279 8 4 32 4 256 4 256 16 256 32 4 256 32 4 256 4 16 256 8
05-380 16 4 32 4 256 4 256 32 4 256 4 256 4 256 128 4 256 4 32 16 4 256 4
06-116 16 4 32 4 256 4 256 32 4 256 4 256 4 256 128 4 256 4 32 32 4 256 4

IPM, imipenem; MEM, meropenem; ATM, aztreonam; CAZ, ceftazidime; CAZ/CLA, ceftazidime/clavulanate; FEP, cefepime; TZP, piperacillin-
tazobactam; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; CIP, ciprofloxacin; LEV, levofloxacin; CHL, chloramphenicol; PO, polymyxin.

FEMS Immunol Med Microbiol 54 (2008) 330–338

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
334 B. Libisch et al.

as donors under the experimental conditions applied. In
case of the Budapest isolates integron B cotransferred with
blaPER-1, while integron A was not mobilized. Altogether,
nine nonduplicate transconjugant colonies were analysed by
PCR and the same results were obtained for all nine
colonies. Plasmid analysis detected an about 40-kb plasmid
in isolates 05-380 and 06-116 but no plasmid DNA was
detectable in the Belgrade isolates. These findings suggest
that blaPER-1 and integron B are located on a conjugative
plasmid in the Budapest isolates but have a chromosomal
localization in the isolates from Serbia.
After incubation at 4 1C for 2 weeks, altogether 150 and
by clavulanate. Boronic acid showed no MIC potentiation
in combination with ceftazidime for any of the isolates
(Table 4).
Experiments designed to examine the impact of a Mex
efflux pump inhibitor on MICs for ciprofloxacin and levo-
floxacin revealed that 50 mg mL1 PAbN could restore sus-
ceptibility to levofloxacin but not to ciprofloxacin for the
PER-1-producing and the cured isolates, both harbouring
an aac(6 0 )-Ib-cr gene. The MIC of PAbN alone for these
isolates was 4 200 mg mL1. The presence of the 40-kb
plasmid had no detectable impact on the fluoroquinolone
resistance of the isolates tested.

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80 colonies of isolates 05-380 and 06-116 were screened,
respectively, for inhibition of growth by ceftazidime at
128 mg mL1. We identified one colony with this phenotype
for both isolates that were designated 05-380CU and
06-116CU and were selected for further characterization.
Both cured isolates displayed serotype O11 and an RAPD
profile identical to that of the respective plasmid-containing
isolate. Plasmid profiling showed that the 40-kb plasmid was
not present in these cured isolates. The loss of plasmid was
concurrent with the loss of the blaPER-1 determinant and also
of integron B from the cells.
Phenotypic characterization of the PER-1-
producing and plasmid-cured P. aeruginosa
isolates and the transconjugants
The results for phenotypic tests on the antibiotic resistance
profile of a selection of PER-1-producing and plasmid-cured
P. aeruginosa isolates and a transconjugant are shown in
Table 4. The transfer of the blaPER-1 determinant and
integron B to the recipient P. putida strain UWC1 conferred
a high level of resistance to ceftazidime in the transconju-
gants that was reversed to susceptibility by clavulanic acid.
On the other hand, the cured isolates carrying the blaOXA-74
gene but not blaPER-1 and blaOXA-2 displayed intermediate
resistance to ceftazidime that was only marginally inhibited
Serotyping and PFGE
All the PER-1-producing P. aeruginosa isolates from Hun-
gary and Serbia belonged to serotype O11. Macrorestriction
analysis revealed a clonal relationship between the four PER-
1-positive isolates from Serbia (PFGE type A, Dice coeffi-
cient Z83%). Isolates 05-176, 05-179 and 05-279 had  1
band difference among each other and thus may belong to
an epidemic clone in the MMA hospital, while isolate 05-
181 displayed three band differences compared with the
other three Belgrade isolates. Therefore, a small cluster of
PER-1-positive infections involving four patients was iden-
tified at the MMA hospital in Belgrade (Table 1). The two
isolates from Hungary displayed 80% similarity to each
other (PFGE type B) and were unrelated by PFGE to the
Belgrade isolates. The PER-1-positive reference isolates
MUL and PER12 (Poirel et al., 2005; Table 5) shared 84%
similarity and clustered to PFGE type C while isolate RNL-1
could not be assigned to any of the above-mentioned PFGE
types.
MLST
Allelic profiles were determined for isolates 05-176 from
Belgrade, 05-380 and 06-116 from Budapest, three

Table 4. Phenotypic studies on selected PER-1-producing and plasmid cured Pseudomonas aeruginosa isolates and a transconjugant
MIC (mg mL1)

Isolate CAZ CAZ/CLA CAZ/BA FEP FEP/CLA CIP CIP/PAbN LEV LEV/PAbN
05-380 4 256 32 4 256 4 256 256 4 32 2 16 0.25
06-116 4 256 32 4 256 4 256 256 4 32 2 32 0.25
05-380CU 16 8 16 4 256 256 4 32 2 16 0.25
05-380TCw 4 256 1 4 256 32 1 0.12 0.064 0.25 0.064
UWC1 2 1 1 0.5 0.5 0.12 0.064 0.25 0.064
Plasmid cured isolate of strain 05-380.
w
Transconjugant Pseudomonas putida obtained by mating the recipient P. putida strain UWC1 with Pseudomonas aeruginosa 05-380 as donor.
CAZ, ceftazidime; CAZ/CLA, ceftazidime/clavulanate; CAZ/BA, ceftazidime plus 200 mg mL1 phenylboronic acid; FEP, cefepime; FEP/CLA, cefepime/
clavulanate; CIP, ciprofloxacin; CIP/PAbN, ciprofloxacin plus 50 mg mL1 phenylalanine arginine b-naphthylamide; LEV, levofloxacin; LEV/PAbN,
levofloxacin plus 50 mg mL1 phenylalanine arginine b-naphthylamide.

c 2008 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 54 (2008) 330–338
Published by Blackwell Publishing Ltd. All rights reserved
PER-1-producing P. aeruginosa from Hungary and Serbia 335

Table 5. Comparison of representative PER-1-producing Pseudomonas aeruginosa isolates from Hungary, Serbia, France and Belgium by serotype,
PFGE and MLST
Allelic numbers

Isolate Serotype PFGE type acsA aroE guaA mutL nouD ppsA trpE STw CCz Town Country
05-176 O11 A1 38 11 3 13 1 2 4 ST235 CC11 Belgrade Serbia
05-380 O11 B1 38 11 3 13 1 2 4 ST235 CC11 Budapest Hungary
06-116 O11 B2 38 11 3 13 1 2 4 ST235 CC11 Budapest Hungary
PER12 O11 C1 38 11 3 13 66 2 4 ST533 CC11 Ghent Belgium
MUL O11 C2 38 93 3 13 1 2 4 ST534 CC11 Mulhouse France
RNL-1 O11 D 5 4 5 5 5 20 4 ST532 S Garches France
Internal fragments of these seven housekeeping genes were sequenced and allelic numbers were assigned using the Pseudomonas aeruginosa

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Multilocus Sequence Typing database (http://pubmlst.org/paeruginosa/) (Curran et al., 2004; Jolley et al., 2004).
w
Each different allelic combination (or profile) is assigned a ST using the MLST database.
z
CC stands for clonal complex, a group of strains sharing Z5 common alleles (Curran et al., 2004; Giske et al., 2006). S stands for singleton.

representative PER-1-producing P. aeruginosa isolates of Discussion

PFGE types A and B (Table 5). All three isolates could be
assigned to ST235, which, according to the eBURST analysis,
is the founder ST of the recently described clonal complex
CC11. ST235 has already been assigned to PER-1-producing
P. aeruginosa isolates from Poland and Turkey (Empel et al.,
2007). We also determined the ST of three reference isolates
from France and Belgium (isolates RNL-1, MUL and
PER12). Isolates PER12 and MUL displayed ST533 and
ST534, respectively, that are single-locus variants (SLVs) of
ST235, namely, they share six identical alleles with ST235
out of the seven sequenced housekeeping genes. Thus, these
two isolates also belong to complex CC11 (Table 5). Finally,
isolate RNL-1 proved unrelated to all the other isolates as it
had a novel ST, ST532 that does not belong to any of the
presently described clonal complexes (Table 5). This finding
is in accordance with the results gained by PFGE.
Clinical data for isolates 06-116 and 05-380
Isolate 06-116 was cultured in 2002 from the nasal swab
sample of a Romanian citizen on admission to the Budapest
hospital; therefore, this patient was a carrier of the PER-1-
producing P. aeruginosa strain. Further clinical data were not
available for this patient.
Isolate 05-380 was cultured at the ICU of the OBSI
hospital, Budapest, from the tracheal aspirate of a polytrau-
matic Hungarian patient within 36 h of admission. This
patient suffered a car accident in Bucharest, Romania 5 days
earlier and was initially treated in a Bucharest hospital where
she underwent surgery, blood transfusion and ceftazidime
antibiotic therapy. After relocation to the Budapest hospital
multidrug-resistant P. aeruginosa was repeatedly cultured
from her tracheal and blood samples. The central venous
catheter-associated bacteraemia was successfully treated by
removing the catheter and by combination therapy using
imipenem and amikacin.
The emergence of PER-1 ESBL-producing Gram-negative
clinical pathogens poses a severe challenge to antimicrobial
chemotherapy with an increasing number of reports world-
wide (Weldhagen et al., 2003; Pagani et al., 2004; Paterson &
Bonomo, 2005; Empel et al., 2007; Strateva et al., 2007).
Earlier works examined the genetic context of the blaPER-1
genes (Mantengoli & Rossolini, 2005; Poirel et al., 2005),
however, available information is scarce on the clonal
relationships of PER-1-producing P. aeruginosa isolates
from different countries.
This is the first report of blaPER-1-positive P. aeruginosa
from Serbia and also of the occurrence of PER-1-producing
P. aeruginosa clinical isolates belonging to the international
clonal complex CC11 from Hungary, Serbia, France and
Belgium. The first PER-1-producing isolates in Eastern
Europe were reported from Poland and Romania (Empel
et al., 2007; Naas et al., 2007), suggesting that this resistance
determinant has a wide geographic distribution in this
region of Europe. Our study revealed that ESBL-producing
P. aeruginosa occur only sporadically in Hungary, with a
prevalence rate of about 1.3% (2/144) among ceftazidime-
nonsusceptible clinical isolates. In contrast to this observa-
tion 12.9% (4/31) of the P. aeruginosa isolates obtained from
the MMA hospital in Belgrade were blaPER-1 positive.
Three novel class 1 integrons were described in this work
and we also identified the aac(6 0 )-Ib-cr gene in PER-1-
producing P. aeruginosa isolates from Hungary. To the best of
our knowledge the aac(6 0 )-Ib-cr fluroquinolone-acetylating
aminoglycoside acetyltransferase gene was detected for the
first time in P. aeruginosa. This gene had been reported
initially from clinical isolates of the Enterobacteriaceae
(Robicsek et al., 2006a, b; Ambrozic Avgustin et al., 2007;
Cordeiro et al., 2007).
The antibiotic resistance profile of the aac(6 0 )-Ib-cr positive
panresistant isolates 05-380 and 06-116 was further investi-
gated using inhibitors for various resistance mechanisms. The

FEMS Immunol Med Microbiol 54 (2008) 330–338

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
336
influence of the Mex efflux inhibitor PAbN was examined on
the ciprofloxacin and levofloxacin MICs of these two Buda-
pest isolates (Table 4). A decrease of more than 16 dilution
steps was detected for both antibiotics; however, susceptibility
was restored by 50 mg mL1 PAbN only for levofloxacin,
suggesting the presence of an additional resistance mechan-
ism for ciprofloxacin. These observations are in accordance
with the substrate profile determined for the aac(6 0 )-Ib-cr
gene that had detectable acetylating activity only with
ciprofloxacin but not with levofloxacin as a substrate
(Robicsek et al., 2006b). Thus, using the efflux inhibitor
PAbN, we could provide an indirect clue for the presence of
the aac(6 0 )-Ib-cr gene in isolates 05-380 and 06-116.
The ceftazidime resistance of isolates 05-380 and 06-116
was only incompletely reversed by clavulanate, similar to the
PER-1- and OXA-17-producing P. aeruginosa isolates from
Turkey and the PER-1- and OXA-74-producing P. aeruginosa
isolates from Poland (Danel et al., 1999; Empel et al., 2007).
Our experiments with ceftazidime/boronic acid combina-
tions suggest that an AmpC-meditated mechanism did not
significantly contribute to the observed ceftazidime MIC
values for isolates 05-380 and 06-116. The plasmid-cured
isolates harbouring blaOXA-74 but not blaPER-1 and blaOXA-2
displayed intermediate resistance to ceftazidime and high-
level resistance to cefepime (Table 4). This phenotype is
similar to that observed for a blaOXA-31-positive clinical
P. aeruginosa isolate from France that remained susceptible
to ceftazidime but had an MIC of 128 mg L1 for cefepime
(Aubert et al., 2001).
It is a worrisome observation that both isolates recovered
in Budapest were resistant to all antibiotics tested including
polymyxin and ceftazidime–clavulanate according to cur-
rent CLSI breakpoints (CLSI, 2008) (Table 3), leaving
limited options for antibiotic therapy. The applied therapy
for the patient infected by isolate 05-380 concurred with
earlier recommendations that the best treatment for a PER-
1-producing P. aeruginosa strain would be imipenem plus
amikacin. The combination of amikacin with imipenem was
shown to be synergistic despite a high amikacin MIC
(Mimoz et al., 1999).
The isolation of PER-1-producing P. aeruginosa from
another patient who had initially been hospitalized abroad
was recently reported from Hungary (Szabó et al., 2008). In
April 2006, a Hungarian tourist was hospitalized because of
his burn and mechanical injuries in Egypt. Five days later, he
was transferred to the Burn Unit of the State Health Center,
Budapest, Hungary. On the day of admission, a PER-1-
producing P. aeruginosa strain was cultured from his burn
wound. It may be proposed that dissemination by human
carriers is an important factor in the emergence of PER-1-
producing P. aeruginosa strains in Hungary.
The blaPER-1-harbouring isolates from Hungary and Ser-
bia shared some common features: all isolates could be


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
B. Libisch et al.
assigned to the serotype O11 and to ST ST235 and carried
integron B (Fig. 1). Also, using MLST it was possible to
establish genetic relatedness between PER-1-producing
P. aeruginosa isolates from Hungary, Serbia, France and
Belgium, as all isolates with the exception of isolate RNL-1
could be assigned to clonal complex CC11.
The criteria developed to determine strain identity by
PFGE are stringent and are not convenient for studies of
large populations of organisms collected over extended
periods of 1 year or longer (Tenover et al., 1995). While
interpreting PFGE banding patterns, we observed slight
differences in levels of genetic similarity between some of
Downloaded from https://academic.oup.com/femspd/article/54/3/330/512859 by guest on 22 October 2020
the isolates as determined by visual inspection vs. computa-
tional analysis (data not shown). The overall classification of
isolates into the different PFGE genotypes was not influ-
enced by these slight differences. Similar findings were also
reported by other authors (Duck et al., 2003; Kawalec et al.,
2007), together with recommendations that there is a need
for an effective standardization and development of the
software packages and applications used for PFGE gel analysis
(Duck et al., 2003). These observations and recommenda-
tions highlight the advantages of the MLST approach in
providing unambiguous interpretation of the experimental
data and in revealing clonal relatedness between isolates
when this is not readily apparent using PFGE (Giske et al.,
2006).
The CC11 clonal lineage has already been detected in nine
European countries including Turkey as well as in the United
States (Giske et al., 2006; Empel et al., 2007; Libisch et al.,
2008 and this work). Our MLST typing of PER-1-producing
P. aeruginosa clinical isolates from four European countries
extends the documented geographical distribution of clonal
complex CC11 and underscores its role in the dissemination
of the blaPER-1 determinant.
Acknowledgements
This work was financially supported by the European Union
through the DRESP2 FP6 grant with no. LSHM-CT-2005-
018705 and by the National Center for Epidemiology. We
thank B. Kovács for technical assistance. This publication
made use of the Pseudomonas aeruginosa Multi Locus
Sequence Typing website (http://pubmlst.org/paeruginosa/)
sited at the University of Oxford. The development of this
site has been funded by the Wellcome Trust.
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