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

Rydzewski K, Schulz T, Brzuszkiewicz E, Holland G, Lück C, Fleischer J, Grunow R, Heuner K - BMC Microbiol. (2014)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Interactions of Bacterial Pathogens, Centre for Biological Threats and Special Pathogens, Division 2 (ZBS 2), Robert Koch Institute, Nordufer 20, Berlin 13353, Germany. heunerk@rki.de.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4230796&req=5

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.

Mentions: These experiments were followed by co-culture studies using macrophage-like cell lines of human (U937) or mice (J774A.1) origin as host cells. We found that strain W12-1067 was able to persist within U937 cells (data not shown), and it replicated intracellularly in J774A.1 cells (Figure 3). Replication within J774A.1 was slower than that of L. pneumophila Paris. However, W12-1067 multiplied about 16-fold within 4 days of co-incubation, with a 24 h lag-phase at the beginning of the infection (Figure 3), indicating that the new isolate is able to infect and multiply within eukaryotic cells. Cells of strain W12-1067 grown in medium T at 37°C possessed a rod-shaped, slightly pleomorphic morphology (Figure 4A). EM analysis of the infection assay revealed that strain W12-1067 was localized intracellularly within a vacuole 96 h after infection (Figure 4B–D). The data indicate that W12-1067 is able to replicate intracellularly, but from EM analysis it was not clear yet whether the bacteria were able to escape from the vacuole. Experiments are under way to investigate this question further.


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

Rydzewski K, Schulz T, Brzuszkiewicz E, Holland G, Lück C, Fleischer J, Grunow R, Heuner K - BMC Microbiol. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230796&req=5

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.
Mentions: These experiments were followed by co-culture studies using macrophage-like cell lines of human (U937) or mice (J774A.1) origin as host cells. We found that strain W12-1067 was able to persist within U937 cells (data not shown), and it replicated intracellularly in J774A.1 cells (Figure 3). Replication within J774A.1 was slower than that of L. pneumophila Paris. However, W12-1067 multiplied about 16-fold within 4 days of co-incubation, with a 24 h lag-phase at the beginning of the infection (Figure 3), indicating that the new isolate is able to infect and multiply within eukaryotic cells. Cells of strain W12-1067 grown in medium T at 37°C possessed a rod-shaped, slightly pleomorphic morphology (Figure 4A). EM analysis of the infection assay revealed that strain W12-1067 was localized intracellularly within a vacuole 96 h after infection (Figure 4B–D). The data indicate that W12-1067 is able to replicate intracellularly, but from EM analysis it was not clear yet whether the bacteria were able to escape from the vacuole. Experiments are under way to investigate this question further.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cellular Interactions of Bacterial Pathogens, Centre for Biological Threats and Special Pathogens, Division 2 (ZBS 2), Robert Koch Institute, Nordufer 20, Berlin 13353, Germany. heunerk@rki.de.

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.

Show MeSH
Related in: MedlinePlus