Isolation and Characterization of Cytotoxic, Aggregative Citrobacter freundii

Abstract

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using Xba I allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 “cytotoxic and aggregative C. freundii .” Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii .

The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

“…C. freundii strain CF74 was isolated from a fecal sample from a goat, as reported previously (26). All strains were grown aerobically at 37°C in Luria-Bertani (LB) medium.…”

“…Comparison of in vitro adhesion to host cells was performed using the human epidermoid carcinoma cell line HEp-2 (CCC0068; Beijing Union Medical College cell resource center), as previously described (26). Infections were performed at a multiplicity of infection (MOI) of 100:1.…”

“…The relative amount of cytotoxicity was expressed as follows: (experimental release Ϫ spontaneous release)/(maximum release Ϫ spontaneous release) ϫ 100, where the spontaneous release was the amount of LDH activity in the supernatant of uninfected cells and the maximum release was that when cells were lysed with the lysis buffer provided by the manufacturer. All experiments were performed two times in duplicate (26).…”

“…Recently, we identified a C. freundii strain, CF74, that showed an aggregative adherence pattern and cytotoxicity to HEp-2 cells. The strain was found to contain a complete T6SS located on a genomic island (GI), and the T6SS gene cluster consists of 16 genes, including key T6SS genes (clpV, hcp-2, and vgr) (26). In this study, we constructed clpV, hcp-2, vgr, and T6SS GI deletion mutants in CF74 and demonstrate that the T6SS affects the transcription of over 84 genes, and especially the flagellar system, at the levels of transcription and translation and is involved in the secretion of FliC.…”

Evaluation of the Virulence Markers in the Clinical Isolates of Citrobacter Species: The First Report from India

“…A recent study which pertained to the virulence markers in Citrobacter freundii was conducted by Li Bai et al, [8] in 2012, who found that the aggregative adherence and cytotoxicity played an important role in diarrhoea which was caused by the same organism. Taylor [9] reviewed that the bacteria are killed by normal human serum through the lytic activity of the alternate complement system.…”