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Genomic characterization of a large outbreak of Legionella pneumophila serogroup 1 strains in Quebec City, 2012.

Lévesque S, Plante PL, Mendis N, Cantin P, Marchand G, Charest H, Raymond F, Huot C, Goupil-Sormany I, Desbiens F, Faucher SP, Corbeil J, Tremblay C - PLoS ONE (2014)

Bottom Line: Two new Legionellaceae plasmids were found only in the epidemic strain.The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates.At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Santé Publique du Québec (LSPQ)/Institut National de Santé Publique du Québec, Québec, Canada.

ABSTRACT
During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire's disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.

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Related in: MedlinePlus

Interaction of L. pneumophila serogroup 1 strains with host cells.One strain from patient (ID119442), one strain from the outbreak source's cooling tower (ID120292), and three from environment (other that the outbreak source) (ID120145, ID120086 and ID120329) were tested. JR32 and an isogenic dotA deficient strain was used as a negative control for intracellular multiplication (ICM) and cytotoxicity. A) ICM in infected human macrophages (THP-1) and Acanthamoeba castellanii (AC). The data represent the ratio between the CFU at time 72 h and the CFU at time 0. Star represent difference statistically significant (P<0.01). B) Evaluation of cytotoxicity on the viability of THP-1 cells after infection for 5 days with the MTT assay. The star represents difference statistically significant (P<0.01). Error bars represent standard deviations derived from three independent experiments.
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pone-0103852-g004: Interaction of L. pneumophila serogroup 1 strains with host cells.One strain from patient (ID119442), one strain from the outbreak source's cooling tower (ID120292), and three from environment (other that the outbreak source) (ID120145, ID120086 and ID120329) were tested. JR32 and an isogenic dotA deficient strain was used as a negative control for intracellular multiplication (ICM) and cytotoxicity. A) ICM in infected human macrophages (THP-1) and Acanthamoeba castellanii (AC). The data represent the ratio between the CFU at time 72 h and the CFU at time 0. Star represent difference statistically significant (P<0.01). B) Evaluation of cytotoxicity on the viability of THP-1 cells after infection for 5 days with the MTT assay. The star represents difference statistically significant (P<0.01). Error bars represent standard deviations derived from three independent experiments.

Mentions: We evaluated the intracellular multiplication in human cultured macrophages (THP-1) and in A. castellanii (AC) and cytotoxicity toward THP-1 cells. The JR32 strain, derived from Philadelphia-1 and the isogenic Icm/Dot deficient dotA mutant was used as a positive and negative control respectively for both assays [22]. As seen in Figure 4A, the outbreak strains (clinical, ID119442 and the environmental source, ID120292) replicated more efficiently in human THP-1 macrophages than in AC (p<0.05). In contrast, environmental strains were equally capable of replicating in THP-1 cells and AC (ID120145, ID120086 and ID120329). The control strain JR32 seems to grow better in THP-1 than AC, but the difference was not significant. There was no difference of replication in AC between the strains tested. The cytotoxicity toward THP-1 cells was investigated by measuring cell viability with the MTT assay. The dose-response curves revealed that the clinical strains were significantly more cytotoxic (p<0.01) than environmental strains toward THP-1 cells at a dose of 10. (Figure 4B).


Genomic characterization of a large outbreak of Legionella pneumophila serogroup 1 strains in Quebec City, 2012.

Lévesque S, Plante PL, Mendis N, Cantin P, Marchand G, Charest H, Raymond F, Huot C, Goupil-Sormany I, Desbiens F, Faucher SP, Corbeil J, Tremblay C - PLoS ONE (2014)

Interaction of L. pneumophila serogroup 1 strains with host cells.One strain from patient (ID119442), one strain from the outbreak source's cooling tower (ID120292), and three from environment (other that the outbreak source) (ID120145, ID120086 and ID120329) were tested. JR32 and an isogenic dotA deficient strain was used as a negative control for intracellular multiplication (ICM) and cytotoxicity. A) ICM in infected human macrophages (THP-1) and Acanthamoeba castellanii (AC). The data represent the ratio between the CFU at time 72 h and the CFU at time 0. Star represent difference statistically significant (P<0.01). B) Evaluation of cytotoxicity on the viability of THP-1 cells after infection for 5 days with the MTT assay. The star represents difference statistically significant (P<0.01). Error bars represent standard deviations derived from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126679&req=5

pone-0103852-g004: Interaction of L. pneumophila serogroup 1 strains with host cells.One strain from patient (ID119442), one strain from the outbreak source's cooling tower (ID120292), and three from environment (other that the outbreak source) (ID120145, ID120086 and ID120329) were tested. JR32 and an isogenic dotA deficient strain was used as a negative control for intracellular multiplication (ICM) and cytotoxicity. A) ICM in infected human macrophages (THP-1) and Acanthamoeba castellanii (AC). The data represent the ratio between the CFU at time 72 h and the CFU at time 0. Star represent difference statistically significant (P<0.01). B) Evaluation of cytotoxicity on the viability of THP-1 cells after infection for 5 days with the MTT assay. The star represents difference statistically significant (P<0.01). Error bars represent standard deviations derived from three independent experiments.
Mentions: We evaluated the intracellular multiplication in human cultured macrophages (THP-1) and in A. castellanii (AC) and cytotoxicity toward THP-1 cells. The JR32 strain, derived from Philadelphia-1 and the isogenic Icm/Dot deficient dotA mutant was used as a positive and negative control respectively for both assays [22]. As seen in Figure 4A, the outbreak strains (clinical, ID119442 and the environmental source, ID120292) replicated more efficiently in human THP-1 macrophages than in AC (p<0.05). In contrast, environmental strains were equally capable of replicating in THP-1 cells and AC (ID120145, ID120086 and ID120329). The control strain JR32 seems to grow better in THP-1 than AC, but the difference was not significant. There was no difference of replication in AC between the strains tested. The cytotoxicity toward THP-1 cells was investigated by measuring cell viability with the MTT assay. The dose-response curves revealed that the clinical strains were significantly more cytotoxic (p<0.01) than environmental strains toward THP-1 cells at a dose of 10. (Figure 4B).

Bottom Line: Two new Legionellaceae plasmids were found only in the epidemic strain.The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates.At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Santé Publique du Québec (LSPQ)/Institut National de Santé Publique du Québec, Québec, Canada.

ABSTRACT
During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire's disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.

Show MeSH
Related in: MedlinePlus