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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- 14-46; Fax: +33-1-49-28-24-72; E-mail: [email protected] Ó The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected] 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 Socie´te´ Fran¸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 1. Lauretti L, Riccio ML, Mazzariol A et al. Cloning and characteriza- VIM gene was present on the larger plasmid (>150 kb) and restriction patterns generated with EcoRI finger- tion of blaVIM, a new integron-borne metallo-b-lactamase gene fromPseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother printing confirmed that VIM-1-encoding plasmids were identical 2. Walsh TR, Toleman MA, Nordmann P. Metallo-b-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: 306–25.
3. Giakoupi P, Xanthaki A, Kanelopoulou M et al. VIM-1 metallo-b- Cloning of genomic DNA fragments allowed the isolation of two lactamase-producing Klebsiella pneumoniae strains in Greek hospitals.
types of clones, one able to grow on imipenem (1 or 2 mg/L) and the J Clin Microbiol 2003; 41: 3893–6.
other able to grow on aztreonam (2 or 4 mg/L). The respective 4. Decre´ D, Gachot B, Lucet JC et al. Clinical and bacteriological phenotypes were consistent with an insert coding for the VIM epidemiology of extended-spectrum b-lactamase producing strains of enzyme in the first clone and an ESBL for the second clone.
Klebsiella pneumoniae in a medical intensive care unit. Clin Infect Dis1998; 27: 834–44.
These results were confirmed by amplification.
5. Decre´ D, Burghoffer B, Gautier V et al. Outbreak of multi-resistant Nucleotide sequence analysis of an 4.5 kb clone carrying Klebsiella oxytoca involving strains with extended-spectrum b-lactamases the MBL determinant revealed that the blaVIM is part of a class 1 and strains with extended-spectrum activity of the chromosomal integron. DNA sequencing showed a gene cassette array that b-lactamase. J Antimicrob Chemother 2004; 54: 881–8.
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 somal DNA restriction patterns produced by pulsed-field gel electro- another aminoglycoside modifying gene, the adenyltransferase phoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: aadA1. This structure was preceded by an integrase 1 gene. Similar cassettes were first reported in P. aeruginosa1 and more recently 7. Livermore DM. b-Lactamases in laboratory and clinical resistance.
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In conclusion, we report the first outbreak of K. pneumoniae 8. Barnaud G, Arlet G, Verdet C et al. Salmonella enteritidis: AmpC strains producing both VIM-1 and SHV-5 in France. The strain was plasmid-mediated inducible b-lactamase (DHA-1) with an ampR gene imported from Greece. Genetic analysis confirmed that the epi- from Morganella morganii. Antimicrob Agents Chemother 1998; 42: demic strain is closely related to one of the epidemic strains isolated in Athens teaching hospitals and that the bla 9. Arakawa Y, Shibata N, Shibayama K et al. Convenient test for screening metallo-b-lactamases-producing Gram-negative bacteria by class I integron similar to those reported in isolates of Entero- using thiol compounds. J Clin Microbiol 2000; 38: 40–3.
10. Kado CI, Lui ST. A rapid procedure for detection and isolation of This study highlights the potential risk of spread of multires- large and small plasmids. J Bacteriol 1981; 145: 1365–73.
istant bacteria with international transfer of patients and confirms 11. Luzzaro F, Docquier JD, Colinon C et al. Emergence in Klebsiella the necessity of systematic screening of these patients at admission.
pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4metallo-b-lactamase encoded by a conjugative plasmid. Antimicrob Agents Chemother 2004; 48: 648–50.
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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