180 Letters to the Editor / International Journal of Antimicrobial Agents 38 (2011) 177–189 and aadA1. Finally, the genetic platforms identified in transconju- gants TcPs, TcPm and TcEc were all identical and were highly similar to the one found in TcKp2 from which they differed only by (i) a deletion in the 3 ′ CS region in TcPs, TcPm and TcEc, and (ii) a trunca- tion in the 5 ′ region of aadA gene found in TcKp2. Thus, the bla VIM-1 gene was integrated in three different types of integrons, which illustrates the diversity of the genetic platforms associated with this gene, as reported previously [3]. One of these integrons was shared by the Ps, Ec and Pm strains and was inserted in a highly conserved plasmid, as judged from comparison of the EcoRI, NdeI and SphI restriction patterns. Moreover, these three strains co-produced the additional ESBL SHV-5, as opposed to Pa and Kp2 that only har- boured bla VIM-1 . Strikingly, the Ec and Pm strains were isolated 133 days after the Ps strain, which was detected 5 days after transfer of the patient to France. Taken altogether, these data strongly suggest that the plasmid harbouring bla VIM-1 was transferred from the Ps strain to the Ec and Pm strains in the digestive tract of the patient. In vivo transfer of -lactamase genes from the Ps strain was assessed in the C3H germ-free mouse model (Anaxem; MICALIS, INRA, Jouy-en-Josas, France). Each mouse was inoculated intragas- trically with the recipient strain (E. coli J53-Azide R ) and 4 days later with the donor strain Ps. Fresh faecal samples were collected once daily, 5 days a week, to follow the evolution of the various bacte- rial populations in mice faeces. Plasmid transfer occurred rapidly (2 days after inoculation with Ps) and was efficient (10 -4 –10 -6 transconjugants per donor). The VIM-1 transconjugants persisted throughout the experiment at a stable level (3.0–4.4 log 10 colony- forming units/g of faeces) without any antimicrobial selective pressure. The bla VIM-1 and bla SHV-5 genes were co-transferred in all transconjugants, which were found to harbour a bla VIM-1 gene inserted in a class 1 integron identical to the one described in TcPs, TcPm and TcEc. Several reports have described the in vivo transfer of - lactamase genes [5], but to our knowledge this is the first demonstration of the in vivo transfer of a carbapenemase deter- minant in an animal model. Even if it cannot be excluded that this in vivo transfer was facilitated by the absence of indigenous flora in this model, this study strongly suggests that the high num- ber of carbapenemase-producing strains in the case reported here was favoured by the in vivo transfer of a single plasmid harbour- ing bla VIM-1 , a mechanism that contributes to the spread of MBLs amongst Enterobacteriaceae and eases their worldwide diffusion. Funding: Grant from the French Ministry of Research (pro- gramme grant UPRES EA1541). The funders had no role in study design, data collection and analysis, decision to publish, or prepa- ration of the manuscript. Competing interests: None declared. Ethical approval: The protocol of the in vivo conjugation assay was approved by the local ad hoc committee. References [1] Galani I, Souli M, Koratzanis E, Koratzanis G, Chryssouli Z, Giamarellou H. Emerg- ing bacterial pathogens: Escherichia coli, Enterobacter aerogenes and Proteus mirabilis clinical isolates harbouring the same transferable plasmid coding for metallo--lactamase VIM-1 in Greece. J Antimicrob Chemother 2007;59:578–9. [2] Psichogiou M, Tassios PT, Avlamis A, Stefanou I, Kosmidis C, Platsouka E, et al. Ongoing epidemic of blaVIM-1-positive Klebsiella pneumoniae in Athens Greece: a prospective survey. J Antimicrob Chemother 2008;61:59–63. [3] Tato M, Coque TM, Baquero F, Cantón R. Dispersal of carbapenemase blaVIM-1 gene associated with different Tn402 variants, mercury transposons, and conjugative plasmids in Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother 2010;54:320–7. [4] Drieux L, Brossier F, Sougakoff W, Jarlier V. Phenotypic detection of extended- spectrum -lactamase production in Enterobacteriaceae: review and bench guide. Clin Microbiol Infect 2008;14(Suppl. 1):90–103. [5] Duval-Iflah Y, Raibaud P, Tancrede C, Rousseau M. R-plasmic transfer from Serratia liquefaciens to Escherichia coli in vitro and in vivo in the digestive tract of gnotobiotic mice associated with human fecal flora. Infect Immun 1980;28:981–90. Laurence Drieux a,b,∗ a Infection Control Team, Hôpital Charles-Foix, F-94200 Ivry-sur-Seine, France b EA 1541, UPMC, Université Pierre et Marie Curie-Paris 6, F-75005 Paris, France Nadège Bourgeois-Nicolaos c,d c EA4065 UFR of Pharmaceutical and Biological Sciences, Université Paris Descartes, F-75006 Paris, France d Laboratory of Bacteriology–Hygiene, Hôpital Antoine Béclère, Université Paris–Sud XI, F 92141 Clamart, France Julie Cremniter Laboratory of Microbiology, Hôpital Ambroise Paré, F-92100 Boulogne-Billancourt, France Christine Lawrence Laboratory of Microbiology, Hôpital Raymond Poincaré, F-92380 Garches, France Vincent Jarlier e,f e EA 1541, UPMC, Université Pierre et Marie Curie-Paris 6, F-75005 Paris, France f Laboratory of Bacteriology–Hygiene, Hôpital Pitié-Salpêtrière, F-75013 Paris, France Florence Doucet-Populaire g,h g Laboratory of Bacteriology–Hygiene, Hôpital Antoine Béclère, Université Paris–Sud XI, F 92141 Clamart, France h Laboratory of Microbiology, Faculté de Pharmacie, Université Paris XI, F-92229 Châtenay-Malabry, France Wladimir Sougakoff i,j,k i EA 1541, UPMC, Université Pierre et Marie Curie-Paris 6, F-75005 Paris, France j Laboratory of Bacteriology–Hygiene, Hôpital Pitié-Salpêtrière, F-75013 Paris, France k INSERM, UMRS-872, LRMA, Site Pitié-Salpêtrière, F-75013 Paris, France ∗ Corresponding author at: Laboratoire de Bactériologie, Faculté Pitié-Salpêtrière, 91 Boulevard de l’Hôpital, F-75634 Paris cedex 13, France. Tel.: +33 1 4077 9745; fax: +33 1 4582 7577. E-mail address: laurence.drieux-rouzet@psl.aphp.fr (L. Drieux) 16 March 2011 doi:10.1016/j.ijantimicag.2011.03.017 Emergence of extended-spectrum -lactamase (ESBL)- producing Klebsiella pneumoniae in Danish hospitals; this is in part explained by spread of two CTX-M-15 clones with multilocus sequence types 15 and 16 in Zealand Sir, Extended-spectrum -lactamase (ESBL)-producing Klebsiella pneumoniae has been increasingly detected in Europe, Africa, Amer- ica and Asia [1]. Before 2007, the occurrence of third-generation cephalosporin (3GC) resistance was low amongst K. pneumoniae isolated from bloodstream infections (BSIs) in Danish patients. However, the rate of resistance has increased from 3.7% in 2006 to 11.2% in 2009 [2]. The aim of the present study was to investigate the spread of 3GC-resistant clinical K. pneumoniae BSI isolates in Denmark by molecular typing. All Danish departments of clinical microbiol- ogy were asked to collect 3GC-resistant (reported as resistant to ceftazidime, ceftriaxone, cefpodoxime or cefotaxime) K. pneumo-