First Detection of VIM-4 Metallo-β-Lactamase-Producing Citrobacter freundii in China

Dear Sir, Carbapenems are one of the most critically important antimicrobials and are considered as last-choice drugs in clinical settings. However, the emergence and dissemination of Enterobacteriaceae carrying acquired metallo-β-lactamases (MBLs) is being increasingly reported throughout the world, presenting a serious threat to the effectiveness of carbapenem treatment [1]. Acquired MBLs, such as VIM, IMP, and NDM, are associated with different mobile elements, and the genes for VIM-type enzymes are carried as mobile gene cassettes inserted in class 1 integrons. In Citrobacter freundii, MBL-mediated resistance is rare and only scattered studies have reported the isolation of a few MBL-producing strains, particularly in the Far East [2]. To our knowledge, there is no report of VIM-producing C. freundii in China. In this study, we describe the first identification of the blaVIM-4 gene in a clinical C. freundii strain isolated from a patient in a hospital in Wuhu, China. The multidrug-resistant C. freundii CF0638 isolate was recovered from the sputum of a 66-year-old man presenting with diabetic foot to a hospital in Wuhu, China, in September 2011. During the period of hospitalization, symptoms of lower respiratory tract infection emerged and this patient received treatment with cefotaxime and ciprofloxacin for 7 days. The effect of the treatment was not satisfactory. The patient continued to be febrile until he was treated with imipenem-cilastatin (3 g/day). Imipenem-cilastatin treatment was continued for a 2-week course, and the symptoms gradually disappeared during the patient’s hospital stay. Susceptibility testing was performed by an agar dilution method according to the CLSI guidelines (2012) [3]. C. freundii CF0638 exhibited high-level resistance to most β-lactam antibiotics tested. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were 4 mg/L and 1 mg/L, respectively (Table 1). Positive results of the modified Hodge test with meropenem disc and double-disc synergy test between carbapenems and EDTA indicated the production of an MBL. CF0638 was subjected to PCR analysis using the respective primer pairs and amplifying conditions for blaIMP and blaVIM, as described previously [4], and followed by DNA sequencing. Nucleotide sequence analysis and homology searches were carried out using the BLAST database (http://www.ncbi.nlm.nih.gov). To determine whether the imipenem resistance was transferable, conjugation experiments were carried out in Luria-Bertani (LB) broth with sodium azide-resistant Escherichia coli J53 as the recipient. Transconjugants were selected on LB agar plates supplemented with sodium azide (100 mg/L) and cefotaxime (4 mg/L). PCR amplification and nucleotide sequence analysis showed that CF0638 harbored blaVIM-4. Conjugation experiments showed that imipenem resistance was successfully transferred to the recipient E. coli J53. PCR analysis of the transconjugant strain was positive for the blaVIM-4 gene. Susceptibility test results of C. freundii CF0638 and its transconjugant are presented in Table 1. C. freundii has been recognized as an opportunistic pathogen that is rarely involved in nosocomial infections. In neonates and