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 XbaI 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.

Figure 1. Dendrogram of the 26 C. freundii isolates based on PFGE patterns.

Shown at the right are PFGE XbaI pattern, isolate name, pulse type, source, and sequence type (ST).

Figure 2. Relationships among the C. freundii isolates as indicated by multi-locus sequence typing data.

A. Neighbor joining tree showing sequence types (STs). For each ST, sources D, H, and A refer to isolates from diarrheal patients, humans of unknown diarrheal status, and animals or insects. O157 refers to serotyping results for O157 antigen. TR refers to tellurite resistance. B. Neighbor net of the STs. Cluster divisions based on neighbor joining tree is marked on both A and B. After ST in square brackets are isolate names.

Figure 3. HEp-2 cell adhesion of C. freundii isolates.

Light micrographs show the adherence patterns displayed by C. freundii strains CF72 and CF74 and control bacterial strains as labeled. The prototype EAEC strain 042, EPEC strain 2348/69, and HB101 displayed aggregative adherence, localized adherence, and non-adherence, respectively. Bar: 10 µm.

Figure 4. HEp-2 cell cytotoxicity of C. freundii isolates.

A: Lactate dehydrogenase (LDH) released by HEp-2 cells after growth with C. freundii isolates. LDH release from HEp-2 cells after exposure to CF72, CF74, and HB101 at different points in time (hours) was measured using optical density reading at A490/630 (vertical axis). B: Morphological changes of HEp-2 cells after growth with C. freundii isolates. Cell rounding, detachment, and death can be seen clearly after 8 h of exposure to C. freundii CF74 in comparison to C. freundii CF72, E coli HB101 and 17-2. Bar: 50 µm.

Figure 5. T6SS in C. freundii CF74.

Here is shown a schematic representation of the C. freundii CF74 T6SS gene cluster compared to the T6SS clusters of Salmonella enterica serovar Agona strain SL483 and enteroaggregative E. coli (EAEC) strain 55989. CF72 has only two T6SS genes present at the same site where CF74 T6SS resides. Conserved T6SS components are highlighted in the same color. The GC content of CF74 T6SS gene clusters is shown at the bottom.

Figure 6. Genetic organization and comparison of fimbriae islands present in CF74.

A. Genetic structure of GI38 and comparison to its homologous fimbrial gene cluster in Salmonella enterica serovar Virchow. The homologous region in CF72 is also shown. B. Genetic structure of GI40 and comparison with its homologous fimbrial gene cluster in Enterobacter aerogenes. Shown below gene bar are locus tags. Gene functions are shown at the top. The relative numbers of between two homologous genes and between the two strains are shown at the protein and nucleotide sequence levels.