Journal of Antimicrobial Chemotherapy (2006) 57, 142–145doi:10.1093/jac/dki389Advance Access publication 10 November 2005
First outbreak of multidrug-resistant Klebsiella pneumoniae carrying
blaVIM-1 and blaSHV-5 in a French university hospital
Najiby Kassis-Chikhani1,2, Dominique Decre´3*, Vale´rie Gautier3, Be´atrice Burghoffer3,
Faouzi Saliba4, Daniele Mathieu1, Didier Samuel4, Denis Castaing4, Jean-Claude Petit3,
1Service de Microbiologie, Hoˆpital Paul Brousse, France; 2Equipe Ope´rationnelle d’Hygie`ne, Hoˆpital Paul
Brousse, France; 3UPRES EA n2392, Faculte´ de Me´decine Saint Antoine, France; 4Centre He´pato-Biliaire,
Hoˆpital Paul Brousse, Assistance Publique—Hoˆpitaux de Paris, France
Received 24 August 2005; returned 26 September 2005; revised and accepted 28 September 2005
Objectives: We studied eight imipenem-resistant isolates of Klebsiella pneumoniae involved in anoutbreak in a French teaching hospital.
Methods: The eight isolates were recovered from clinical specimens or rectal swabs. Antibiotic suscept-ibilities were determined using standard agar diffusion and dilution methods including synergy tests.
PFGE was used to study the relatedness of isolates. Genes encoding b-lactamases were characterizedby transfer assays, specific amplification and cloning.
Results: The eight isolates were closely related by PFGE analysis and highly related to a K. pneumoniaestrain from Greece. They were highly resistant to b-lactams, including aztreonam and imipenem (MIC‡32 mg/L), and were positive by the imipenem-EDTA disc synergy test. Isolates were also resistant toaminoglycosides, newer quinolones and sulfamethoxazole, and showed an intermediate level of resistanceto tetracycline. VIM-1 and SHV-5 b-lactamases were revealed in all isolates by PCR. The analysis of plasmidcontents of Escherichia coli DH10B electroporants expressing the VIM-1 b-lactamase or the SHV-5b-lactamase confirmed that the two enzymes were coded by two different plasmids. The blaVIM-1 genewas part of a class 1 integron that also included aac6, dhfrI and aadA genes and was similar to thosereported from strains isolated in Greece.
Conclusions: This study confirms the potential risk of spread of multiresistant bacteria with interna-tional transfer of patients.
Keywords: imipenem resistance, class I integrons, liver transplants
of Pseudomonas aeruginosa in Verona, Italy.1 VIM-typeb-lactamases have been described in various geographical areas2
The emergence of acquired metallo-b-lactamases (MBLs) in
and have been reported in several enterobacterial species but
Gram-negative bacilli is becoming a therapeutic challenge because
P. aeruginosa remains the most important known reservoir of
these b-lactamases possess a broad hydrolysis spectrum that
these enzymes.2 Outbreaks of the VIM-type MBLs have been
includes virtually all b-lactams, except the monobactam aztreo-
reported mostly in P. aeruginosa, but also recently in Klebsiella
nam. IMP- and VIM-type enzymes are the two major types
of MBLs reported. Most acquired MBL genes that have been
We report the first outbreak of colonizations and infections due
reported were inserted on mobile elements (especially integron-
to a K. pneumoniae strain producing VIM-1 MBL in a French
borne gene cassettes). The first member of the VIM-family
University Hospital, which followed transfer of a patient from
determinants, VIM-1, was identified from a clinical isolate
*Correspondence address. Service de Bacte´riologie, Faculte´ de Me´decine Saint Antoine, 27 rue Chaligny 75012 Paris, France. Tel: +33-1-40-01-
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First outbreak of multidrug-resistant Klebsiella pneumoniae
DNA fragments obtained from Sau3A partially digested genomicDNA were ligated into the vector pACYC184 digested with
Imipenem-resistant K. pneumoniae isolates were recovered from
BamH1. E. coli DH10 transformants were selected on Mueller–
patients hospitalized at the Hepatobiliary Surgical Centre of the
Hinton agar supplemented with 1 and 4 mg/L of imipenem or 2 and
Paul Brousse Hospital. It is an 81 bed centre including 15 ICU
8 mg/L of aztreonam. The inserted DNA fragments were sequenced
beds, and admits 500 patients per year, of whom 15% are referred
from outside of France. Since 2002, screening of extended-spectrumb-lactamase (ESBL)-producing strains of Gram-negative bacilli
using rectal swabs has been instituted for all patients admitted tothe ICU.4
The nucleotide sequence of the integron described in this paperhas
One hundred and sixteen imipenem- and aztreonam-resistant isolatesof K. pneumoniae were recovered from eight patients (68 strains
from clinical specimens and 48 from rectal swabs). As all isolatesexhibited the same resistance pattern, the first isolate from each patient
was included in the study. Three imipenem-resistant strains of
K. pneumoniae isolated in Greece (K1, K5 and K8)3 were used ascomparators in the typing methods.
The outbreak of imipenem-resistant K. pneumoniae occurred in thesurgical centre during a 6 month period. The index case (Patient M)was a patient transferred from Greece for fulminant hepatitis and
Clonal relation between isolates by PFGE analysis
the strain of K. pneumoniae was detected at admission. This patient
Genomic DNA, prepared as described previously5 and digested with
was carrying the multiresistant strain in his digestive flora and was
XbaI (Ozyme, New England Biolabs Inc., Saint Quentin en Yvelines,
not infected. The outbreak included the spread of the strain to seven
France) was subjected to PFGE with the CHEF DRIII device (Bio-
other patients (Table 1). The last patient (K) was detected after
Rad). The resulting restriction patterns were interpreted as reported by
PFGE analysis identified one major profile with three subtypes
(Ia, Ib and Ic) (Figure 1). The Ic subtype (patients V and S) showed
Antibiotic susceptibility and synergy testing
four banding differences. Comparison of the PFGE profiles withthose three strains isolated in Greece indicated that our epidemic
Agar dilution and disc diffusion tests were performed according
strain was identical with the strain K5 reported in Athens teaching
to the recommendations of the Comite´ de l’Antibiogramme de la
caise de Microbiologie ClassA extended-spectrum and plasmid-mediated class C b-lactamaseswere detected using synergy tests7,8 To
Antibiotic susceptibility and synergy testing and
detect MBL production, a synergy test using imipenem and EDTA-
containing discs and Etest (AES Laboratoire) were used.9
Identification performed by using the API20E gave identicalresults except for isolates recovered from two patients (S and V)
PCR amplification and molecular characterization of
that were lysine-decarboxylase negative. These two isolates cor-
responded to the subtype Ic in PFGE. All isolates were highly
resistant to b-lactams (>64 mg/L), including aztreonam, and
TEM, -SHV, -CTX-M, -CMY, -VIM, -IMP, -SMP, -ACC, -FOX spe-
cific primers5 and subsequent sequencing of PCR products were
were resistant to imipenem (MICs ‡32 mg/L). All isolates were
positive by the EDTA disc synergy test indicating the presence ofan MBL.
PCR using the primers VIM-1 up and VIM-1 low yielded a
261 bp amplification product that suggested the presence of a
Conjugation experiments were carried out between each test isolate
blaVIM gene, which was confirmed by sequencing to encode
and Escherichia coli K12 J53-2 (met pro RifR) in broth medium.
VIM-1 MBL. The PCR-based screening for ESBL revealed the
Transfer of resistance by electroporation was performed with plasmid
presence of a SHV-type b-lactamase, identified by sequencing as
DNAs (Qiagen Midi Kit, Coutaboeuf, France) transformed into E. coli
SHV-5. Luzzaro et al.11 identified both SHV-12 and VIM-4 in two
DH10B (Invitrogen SARL, Cergy Pontoise, France) by electroporation
strains of Enterobacter cloacae. Scoulica et al.12 reported the
(Bio-Rad). Transconjugants or electroporants were selected on
spread of E. coli strains producing both VIM-1 and a CTX-M-
rifampicin (256 mg/L) and either imipenem (1, 2 or 4 mg/L) or aztreo-
type b-lactamase and recently Galani et al.13 identified the pres-
ence of the b-lactamases VIM-2 and IBC-1 in a strain of E. coli.
Antimicrobial phenotype to other antibiotics showed resistance
to almost all aminoglycosides (MICs for gentamicin of 8 mg/L),
Plasmid DNA was extracted by the alkaline lysis method.10 Plasmid
newer quinolones (MICs of ciprofloxacin >128 mg/L) and the
DNA was purified from electroporant cells with the Qiagen Plasmid
combination of sulfamethoxazole and trimethoprim. Isolates
Midi Kit (Qiagen). For fingerprinting analysis, plasmid DNA was
showed an intermediate resistance level to tetracycline with
Table 1. Characteristics of patients colonized and/or infected with imipenem-resistant K. pneumoniae
Liver transplantation 12.02.03 Rectal swab
Liver transplantation 02.15.04 Blood culture Surgical ward
Liver transplantation 03.11.04 Tracheal fluid Surgical ward
Liver transplantation 04.01.04 Urine culture
Liver transplantation 05.28.04 Blood culture ICU
Liver transplantation 06.17.04 Rectal swab
Liver transplantation 08.06.04 Rectal swab
ATM, aztreonam; IPM, imipenem; CLA, clavulanic acid at 2 mg/L; TZB, tazobactam at 4 mg/L.
aMICs of IMP + EDTA determined by the Etest procedure.
bPatient detected in August during the follow up.
Figure 1. PFGE. Lanes 1–3, PFGE profiles of non-related strains of K. pneumoniae; lane 4, strain K1 from Greece; lane 5, strain K5 from Greece; lane 6, strain K8from Greece; lane 7, isolate M; lane 8, isolate F; lane 9, isolate L1; lane 10, isolate G; lane 11, isolate V; lane 12, isolate S; lane 13, isolate L2; lanes 14, isolate K.
DH10B electroporants selected on imipenem showed an MBL
Transfer of b-lactam resistance and plasmid analysis
phenotype (e.g. resistant to all b-lactams with the exception of
Transfer by conjugation was achieved for only one isolate
aztreonam) while those selected on aztreonam corresponded to
(Patient F) on selective agar containing aztreonam, and the
E. coli J53 transconjugant displayed a typical ESBL phenotype.
Plasmid analysis showed that all isolates harboured three plas-
As reported for VIM-1 and VIM-2 b-lactamases, imipenem resist-
mids of estimated sizes of 100, 130 and >150 kb. The analysis of the
ance was not transferred by conjugation suggesting that the cor-
plasmid contents of E. coli DH10B electroporants expressing the
responding gene is not located on a transferable plasmid.3,13 For the
MBL or those expressing the ESBL in comparison with parental
other isolates the transfer was obtained by electroporation. E. coli
strains indicated that the two b-lactamases were encoded by two
First outbreak of multidrug-resistant Klebsiella pneumoniae
different plasmids. PCR using primers specific for VIM and
SHV genes performed on each electroporant confirmed theseresults. The bla
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the MBL determinant revealed that the blaVIM is part of a class 1
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included the blaVIM determinant, a 60N-aminoglycoside acetyl-
6. Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromo-
transferase aac(60)-Ib gene cassette, the dhfrI determinant and
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In conclusion, we report the first outbreak of K. pneumoniae
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strains producing both VIM-1 and SHV-5 in France. The strain was
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imported from Greece. Genetic analysis confirmed that the epi-
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demic strain is closely related to one of the epidemic strains isolated
in Athens teaching hospitals and that the bla
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12. Soulica EV, Neonakis IK, Gikas AI et al. Spread of blaVIM-producing
We thank Professor Vatopoulos (University of Athens) for provid-
E. coli in a university hospital in Greece. Genetic analysis of the
ing strains K1, K5 and K8. This study was supported by grant
integron carrying the blaVIM-1 metallo-b-lactamase gene. Diag Microbiol
no. EA2392 from the Unite´ Propre de Recherche de l’Enseignement
Supe´rieur (UPRES) and by the European Community (6th PCRD,
13. Galani I, Souli M, Chryssouli Z et al. First identification of an
Escherichia coli clinical isolate producing both metallo-b-lactamase
VIM-2 and extended-spectrum b-lactamase IBC-1. Clin Microbiol Infect2004; 10: 757–9.
14. Miriagou V, Tzelepi E, Gianneli D et al. Escherichia coli with a
self-transferable, multiresistant plasmid coding for metallo-b-lactamase
No declarations were made by the authors of this paper.
VIM-1. Antimicrob Agents Chemother 2003; 47: 395–7.
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