Genetic characterization of atypical Citrobacter freundii

Abstract

The ability of a bacterial population to survive in different niches, as well as in stressful and rapidly changing environmental conditions, depends greatly on its genetic content. To survive such fluctuating conditions, bacteria have evolved different mechanisms to modulate phenotypic variations and related strategies to produce high levels of genetic diversity. Laboratories working in microbiological diagnosis have shown that Citrobacter freundii is very versatile in its colony morphology, as well as in its biochemical, antigenic and pathogenic behaviours. This phenotypic versatility has made C. freundii difficult to identify and it is frequently confused with both Salmonella enterica and Escherichia coli. In order to determine the genomic events and to explain the mechanisms involved in this plasticity, six C. freundii isolates were selected from a phenotypic variation study. An I-CeuI genomic cleavage map was created and eight housekeeping genes, including 16S rRNA, were sequenced. In general, the results showed a range of both phenotypes and genotypes among the isolates with some revealing a greater similarity to C. freundii and some to S. enterica, while others were identified as phenotypic and genotypic intermediary states between the two species. The occurrence of these events in natural populations may have important implications for genomic diversification in bacterial evolution, especially when considering bacterial species boundaries. In addition, such events may have a profound impact on medical science in terms of treatment, course and outcomes of infectious diseases, evading the immune response, and understanding host-pathogen interactions.

Figure 1. Bacterial lineages derived from the phenotypic diversification study.

Shows the lineages A and B derived from the Citrobacter freundii FMU108327/P, each dot represents a selected colony. The pink dots indicate a typical C. freundii biochemical phenotype; yellow dots indicate a “Salmonella-like” biochemical phenotype; the dots in gradient of pink to yellow represent the intermediates biochemical phenotypes. The solid lines indicate the number of colonies selected after each selective round. The parental strain was Citrobacter freundii FMU108327/P, and after the first selection two descendants were generated, which were used to found the next selection, which led to six descendants (in both lineages) being selected, and all of these isolates were used to found the 3^th selection round. This procedure was repeated throughout the 10 selections. The numbered dots P, A₁, A₁₀, B_1, B₂ and B₁₀ indicate the isolates selected for this study.

Figure 2. Pulsed-field gel electrophoresis profiles of l-CeuI restriction fragments from C. freundii isolates.

a) Pulsed-field gel shows CeuI profiles: line 1, C. freundii E9750 NCTC; lane 2, FMU108327/P; lane 3, FMU108327/A₁₀ and lane 4; S. Typhimurium LT2 ATCC 700720. The results of Southern blotting analysis appear with capital letters. These indicate the Ceús individual fragments of C. freundii corresponding to those of S. Typhimurium LT2; b) chromosome sizes and molecular weights of individual CeuI fragments. It also shows the location of every marker gene according to its correspondence in each CeuI fragment. The CeuI-C and CeuI-F fragments of C. freundii were determined by probing the isolated DNA from S. Typhimurium LT2 CeuI-C and CeuI-F fragments; c) the gel from panel a were blotted to N+nylon membranes and probed as follows: lanes 1–4 metH (Table S1); lanes 5–8 DNA from C. freundii fragment CeuI-H and lanes 9–12, metA. CeuI fragments identified by the probes are indicated inside the images with capital letters.

Figure 3. Individual and concatenated maximum likelihood trees.

All of the trees were constructed in Mega 5.01 for a) adk, b) recA, c) gyrB, d) icd, e) purA, f) aph, g) gnd, h) mdh and i) concatenated genes. The assigned names of the genus in which these alleles occur are shown. The scale bar represents the number of substitutions per site.

Figure 4. Proportion of ancestry inferred by STRUCTURE.

Results of the STRUCTURE analysis assuming 4 populations, a) every vertical line represents each of the 49 isolates and indicates the proportion of ancestry from the 4 ancestral populations, using the following colors: green for E. coli, blue for Enterobacter spp. and Klebsiella spp., purple for Citrobacter spp. and red for Salmonella spp.; b) It shows the proportion from Citrobacter and Salmonella ancestries in our isolates, as well as their biochemical phenotypes. It is interesting to highlight the A10 and B2 isolates. In spite of the fact that A10 derivative isolate has 60% identity with Citrobacter ssp., it displayed Salmonella's biochemical phenotype which has remained stable until now; in addition we have the B2 derivative isolate which had a genotype with a majority of identity to Salmonella ssp genus and its biochemical phenotype was variable, thus preventing establishing some biochemical identity.

Figure 5. Mutation frequencies.

Mutation frequencies for each of the 30 positions in 8 genes of 5 strains are shown. The isolates with the greatest number of mutations were B₂ and B_10, which presented the most diversified genotype, and the genes with the greatest number of mutations were adk, icd and recA.