Identification of ACT-1 Plasmid-Mediated AmpC β-Lactamase Producing Citrobacter freundii from a Chinese Patient

Dear Sir, Enterobacteriaceae, other than Escherichia spp. and Klebsiella spp., can cause serious nosocomial infections and appear to have become increasingly drug resistant. Citrobacter freundii, belonging to the Enterobacteriaceae family, is a prominent opportunistic pathogen responsible for serious infections in immunocompromised individuals. C. freundii is characterized by the presence of chromosomally-encoded AmpC β-lactamases and possesses the ability to develop resistance on exposure to broadspectrum cephalosporins [1]. Resistance to third-generation cephalosporins in C. freundii has been attributed to the production of extended-spectrum β-lactamases (ESBLs), but resistance mediated by plasmid-mediated AmpC β-lactamase (pAmpCs) has not been reported to the best of our knowledge. A specific C. freundii, designated CF0513, was isolated from the wound drainage fluid of a 75-yr-old man who suffered from chest trauma and was admitted to a hospital in Wuhu, China, in December 2008. During the period of hospitalization, symptoms of lower respiratory tract infection emerged. This patient was treated with cefotaxime for 7 days, but the treatment was not found to be effective The patient was then administered imipenem-cilastatin (500 mg intravenous, q8 hr) for a period of 7 days, and the symptoms of lower respiratory tract infection gradually disappeared during his hospitalization. The bacterial species identification was performed using the Vitek 2 system (BioMérieux, Marcy l’Etoile, France) and confirmed using API 20E identification kit (BioMérieux, Marcy l’Etoile, France). CF0513 was found to be resistant to all the β-lactams (except carbapenems), and to ciprofloxacin. Further, the minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by the agar dilution method, and the susceptibility data were interpreted in accordance with the recommendations of the Clinical Laboratory Standards Institute, 2012 [2]. CF0513 showed elevated MICs for all β-lactams, except for imipenem and meropenem (<0.25 mg/L), and it was also resistant to ciprofloxacin, levofloxacin, gatifloxacin, gentamicin, and amikacin (Table 1). The presence of AmpC was determined by a modified 3-dimensional test [3]. Plasmid DNA was extracted using a Qiagen Plasmid Purification kit (QIAGEN, Hilden, Germany), and ampC gene amplification was carried out by multiplex PCR using plasmid DNA as a template [4]. The association with mobile elements (IntI1, ISCR1, and ISEcp1) and other resistance genes (qnr, aac(6’)-Ib-cr, qepA, blaTEM, blaSHV, blaCTX-M; and 16S rRNA methylase genes such as armA, rmtA, rmtB, rmtC, rmtD, and rmtE) was investigated by PCR amplification [5-8]. The PCR assay showed that CF0513 carried blaACT-1. In addition, CF0513 coharbored blaCTX-M-14, armA, ISEcp1, and IntI1 (Table 1). DNA sequence analysis showed that blaACT-1 was associated with an ISEcp1 mobile element downstream.