Genome sequence and phenotypic analysis of a first German Francisella sp. isolate (W12-1067) not belonging to the species Francisella tularensis

Abstract

Background: Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany.

Results: We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4-5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (~1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067.

Conclusions: Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany.

Figure 1

Phylogenetic tree analysis. (A) Phylogenetic tree analysis of different Francisella strains using 16S rDNA or (B) 6-loci concatenated DNA sequences. Name and function of genes used for the 6-loci concatenated sequences are given in Table 2. *, 16S rDNA sequences of different isolates of F. guangzhouensis published by [20]; #, Concatenated sequence of L. pneumophila Paris (Lpp) did not exhibit a fopA gene, because no homolog of this gene is present within the genome sequence. Fhol-OSU18, Ft. holartica strain OSU18; Ftul-SchuS4, Ft. tularensis strain SchuS4; Fmed-FSC147, F. mediasiatica strain FSC147, Fnov-U112, Ft. novicida strain U112; Fhis-3523, F. hispaniensis (Ft. novicida-like strain 3523); Fnoa-Toba04, F. noatunensis strain Toba04; Fphi-25015 and 25017, F. philomiragia strain ATCC 25015 and ATCC 25017; F-TX077308, Francisella isolate TX077308.

Figure 2

Growth of different Francisella strains at 25, 30 and 37°C in medium T. (A) Comparison of growth of Francisella isolate W12-1067 at different growth temperatures. (B–D) Comparison of growth of Francisella isolate W12-1067 with the growth of strains Ft. novicida U112 (Ft. novicida) and F. philomiragia 25015 (F. philomiragia ATCC 25015) at 25°C (B), 30°C (C) and 37°C (D). Results are mean standard deviations of three independent experiments of duplicate samples.

Figure 3

Infection assays of Francisella strain W12-1067 and L. pneumophila Paris (Lpp) using the J774A.1 mouse cell line. Growth curve over a period of four days. Cells were infected with bacteria at an MOI of 10. Cells were washed three times with RPMI and incubated with 50 μg/ml of Gentamycin for 1 h to kill extracellular bacteria. Cells were washed again three times with RPMI and covered with 1 ml of RPMI + 10% FCS. The number of CFU per well was determined by plating on CHA agar plates. Results are mean standard deviations of three independent experiments of duplicate samples.

Figure 4

Thin-section EM of Francisella strain W12-1067. (A) Bacteria cultivated in medium T at 37°C possess a rod-shaped, slightly pleomorphic morphology. (B-D) Incubation of J774A.1 cells with bacteria (MOI 10). (B) Overview of host cells with two compartments containing bacteria (96 h post infection). Rectangles mark regions shown at higher magnification in C and D, respectively. (C) Two bacteria in a membrane-bound compartment. (D) Several bacteria in a compartment which shows a clearly discernible membrane at least in some regions.

Figure 5

Genetic organization of T6 secretion systems in Francisella. Organization of the FPI island of Ft. novicida U112 (A) and of genomic islands I (B) and II (C) encoding putative T6-like secretion systems of Francisella isolate W12-1067. Genomic island I is integrated between genes pyrD and tyrA in W12-1067 and Ft. novicida U112, whereas it is integrated between F7308_1884 und F7308_1920 in Francisella strain TX077308 (F-TX077308). Genomic island II is integrated between rpsU and glmS in W12-1067 and is not present in Ft. novicida U112 and Francisella strain TX077308. Genes (ORFs) are indicated by arrows. Gene names are given below the genes, and the protein-encoding gene (peg) numbers are given in Table 5. Genes encoding homologous proteins are boxed in the same color, with the exception of pink (mobile elements) and black (conserved core genome genes).