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 broad-spectrum 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, bla_TEM, bla_SHV, bla_CTX-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 bla_ACT-1. In addition, CF0513 coharbored bla_CTX-M-14, armA, ISEcp1, and IntI1 (Table 1). DNA sequence analysis showed that bla_ACT-1 was associated with an ISEcp1 mobile element downstream.

Plasmid analysis revealed that CF0513 only harbored a 50 kb plasmid. Escherichia coli V517 was used as a reference marker. To determine whether the AmpC phenotype 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 (2 mg/L). The AmpC phenotype was successfully transferred to E. coli J53. The transconjugant plasmids showing an AmpC phenotype were classified according to their incompatibility group, using the PCR replicon-typing scheme described by Carattoli et al. [9]. The plasmid profile revealed that the CF0513 transconjugate carried a bla_ACT-1-positive plasmid that belonged to the IncL/M incompatibility type. The plasmid was also the same size as that of the original strain CF0513. To assess if plasmid p0513, encoding bla_ACT-1, was stable, both the donors and transconjugants were serially split and regrown up to 10 times on separate LB agar plates with cefotaxime (8 mg/L). The plasmid persisted even after the last generation, which indicates that p0513 is highly stable.

A wide variety of plasmid-mediated β-lactamases, especially ESBLs and pAmpCs, have been identified worldwide. However, other than Escherichia spp. and Klebsiella spp., they are rarely investigated in China among Enterobacteriaceae where the resistance problem has increased dramatically. C. freundii, which belongs to the Enterobacteriaceae family, is a prominent opportunistic pathogen and characterized by chromosomally-encoded AmpC β-lactamases. To our knowledge, there are no reports of resistance caused by plasmid-mediated AmpC β-lactamases in C. freundii. In our study, we describe an isolated C. freundii carrying the ACT-1 type pAmpCs. Furthermore, the bla_ACT-1 gene was carried by an IncL/M plasmid and was associated with bla_CTX-M-14, armA, ISEcp1, and IntI1. Due to these characteristics, C. freundii CF0513 was also resistant to aminoglycosides, such as gentamicin and amikacin. To our knowledge, this is the first report of an isolated C. freundii producing ACT-1 AmpC β-lactamase.

In conclusion, the bla_ACT-1 gene associated with ISEcp1 found on a transmissible plasmid in C. freundii is cause for serious concern, as this insertion element facilitates the mobilization and expression of ACT-1-mediated resistance. This is alarming, since horizontal transfer played a role in the dissemination of these multidrug resistant isolates. Such strains, with acquired resistance, further limit the therapeutic options in clinical work. Further investigation of the association between mobile elements and resistance genes is warranted.