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Gene flow in environmental Legionella pneumophila leads to genetic and pathogenic heterogeneity within a Legionnaires' disease outbreak.

McAdam PR, Vander Broek CW, Lindsay DS, Ward MJ, Hanson MF, Gillies M, Watson M, Stevens JM, Edwards GF, Fitzgerald JR - Genome Biol. (2014)

Bottom Line: In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution.Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Legionnaires' disease is a severe form of pneumonia caused by the environmental bacterium Legionella pneumophila. Outbreaks commonly affect people with known risk factors, but the genetic and pathogenic complexity of L. pneumophila within an outbreak is not well understood. Here, we investigate the etiology of the major Legionnaires' disease outbreak that occurred in Edinburgh, UK, in 2012, by examining the evolutionary history, genome content, and virulence of L. pneumophila clinical isolates.

Results: Our high resolution genomic approach reveals that the outbreak was caused by multiple genetic subtypes of L. pneumophila, the majority of which had diversified from a single progenitor through mutation, recombination, and horizontal gene transfer within an environmental reservoir prior to release. In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution. Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.

Conclusions: Taken together, our study indicates previously cryptic levels of pathogen heterogeneity within a Legionnaires' disease outbreak, a discovery that impacts on source attribution for future outbreak investigations. Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

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ST191 outbreak isolates are represented by multiple genetic subtypes which arose by mutation, recombination and horizontal gene transfer. (a) Genome distribution of mutations and predicted recombinant regions among ST191 outbreak isolates. Polymorphisms are mapped to the reference strain Corby. SNPs are represented by short black lines, and variant T4SSs are indicated by colored circles; Dot/Icm T4BSS (variant 1), dark green; Dot/Icm T4BSS (variant 2), light green; Lvh T4ASS (Philadelphia), red; Lvh T4ASS (novel), blue. Predicted recombinant regions are flanked by dashed red lines. (b) Maximum likelihood un-rooted radial phylogeny reconstructed using the non-recombinant core genome of ST191 outbreak isolates. For each node, maximum likelihood bootstrap values are displayed. Filled triangles indicate multiple isolates that were obtained from a single patient as follows; patient 1, yellow; patient 2, red; patient 12, green; patient 13, blue. Presence or absence of genomic regions encoding T4SS is indicated by colored filled circles as per (a).
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Fig2: ST191 outbreak isolates are represented by multiple genetic subtypes which arose by mutation, recombination and horizontal gene transfer. (a) Genome distribution of mutations and predicted recombinant regions among ST191 outbreak isolates. Polymorphisms are mapped to the reference strain Corby. SNPs are represented by short black lines, and variant T4SSs are indicated by colored circles; Dot/Icm T4BSS (variant 1), dark green; Dot/Icm T4BSS (variant 2), light green; Lvh T4ASS (Philadelphia), red; Lvh T4ASS (novel), blue. Predicted recombinant regions are flanked by dashed red lines. (b) Maximum likelihood un-rooted radial phylogeny reconstructed using the non-recombinant core genome of ST191 outbreak isolates. For each node, maximum likelihood bootstrap values are displayed. Filled triangles indicate multiple isolates that were obtained from a single patient as follows; patient 1, yellow; patient 2, red; patient 12, green; patient 13, blue. Presence or absence of genomic regions encoding T4SS is indicated by colored filled circles as per (a).

Mentions: In order to elucidate the relatedness of the 21 ST191 outbreak isolates to each other, the core genome variation among the ST191 outbreak isolates only was examined. Inspection of the sequence alignment revealed the presence of three regions of high SNP density in each of three isolates (12_4480, 12_4499, 12_5054), which were suggestive of recombination events in those strains (Figure 2). Removal of these genomic regions yielded an alignment of 2,694,741 bp, with a total of 42 polymorphic sites (Figure 2). Phylogenetic reconstruction using maximum likelihood and Bayesian methods revealed four distinct subtypes (A to D) among the ST191 outbreak isolates from 15 patients (Figure 2, Table 1). Of note, for patients 10, 11, and 15 the multiple isolates obtained from each were identical, suggesting that the short incubation periods did not support extensive within-host diversification. However, isolates from patient 1 were represented by multiple genetic subtypes of ST191 (clades A and C), which were differentiated by 20 core genome SNPs (Figure 2). Of the four subtypes, three were identified in multiple patients, including subtype A in four patients, subtype B in seven patients, and subtype C in two patients, indicating their wide distribution among patients infected during the outbreak (Table 1). The short timescale between exposure to and isolation of the pathogen during the outbreak and the lack of person-to-person transmission for L. pneumophila [33,34] strongly suggest that the genetic subtypes of ST191 existed in the outbreak source prior to release and evolved from a recent progenitor within the water reservoir by a combination of gene mutation and recombination.Figure 2


Gene flow in environmental Legionella pneumophila leads to genetic and pathogenic heterogeneity within a Legionnaires' disease outbreak.

McAdam PR, Vander Broek CW, Lindsay DS, Ward MJ, Hanson MF, Gillies M, Watson M, Stevens JM, Edwards GF, Fitzgerald JR - Genome Biol. (2014)

ST191 outbreak isolates are represented by multiple genetic subtypes which arose by mutation, recombination and horizontal gene transfer. (a) Genome distribution of mutations and predicted recombinant regions among ST191 outbreak isolates. Polymorphisms are mapped to the reference strain Corby. SNPs are represented by short black lines, and variant T4SSs are indicated by colored circles; Dot/Icm T4BSS (variant 1), dark green; Dot/Icm T4BSS (variant 2), light green; Lvh T4ASS (Philadelphia), red; Lvh T4ASS (novel), blue. Predicted recombinant regions are flanked by dashed red lines. (b) Maximum likelihood un-rooted radial phylogeny reconstructed using the non-recombinant core genome of ST191 outbreak isolates. For each node, maximum likelihood bootstrap values are displayed. Filled triangles indicate multiple isolates that were obtained from a single patient as follows; patient 1, yellow; patient 2, red; patient 12, green; patient 13, blue. Presence or absence of genomic regions encoding T4SS is indicated by colored filled circles as per (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4256819&req=5

Fig2: ST191 outbreak isolates are represented by multiple genetic subtypes which arose by mutation, recombination and horizontal gene transfer. (a) Genome distribution of mutations and predicted recombinant regions among ST191 outbreak isolates. Polymorphisms are mapped to the reference strain Corby. SNPs are represented by short black lines, and variant T4SSs are indicated by colored circles; Dot/Icm T4BSS (variant 1), dark green; Dot/Icm T4BSS (variant 2), light green; Lvh T4ASS (Philadelphia), red; Lvh T4ASS (novel), blue. Predicted recombinant regions are flanked by dashed red lines. (b) Maximum likelihood un-rooted radial phylogeny reconstructed using the non-recombinant core genome of ST191 outbreak isolates. For each node, maximum likelihood bootstrap values are displayed. Filled triangles indicate multiple isolates that were obtained from a single patient as follows; patient 1, yellow; patient 2, red; patient 12, green; patient 13, blue. Presence or absence of genomic regions encoding T4SS is indicated by colored filled circles as per (a).
Mentions: In order to elucidate the relatedness of the 21 ST191 outbreak isolates to each other, the core genome variation among the ST191 outbreak isolates only was examined. Inspection of the sequence alignment revealed the presence of three regions of high SNP density in each of three isolates (12_4480, 12_4499, 12_5054), which were suggestive of recombination events in those strains (Figure 2). Removal of these genomic regions yielded an alignment of 2,694,741 bp, with a total of 42 polymorphic sites (Figure 2). Phylogenetic reconstruction using maximum likelihood and Bayesian methods revealed four distinct subtypes (A to D) among the ST191 outbreak isolates from 15 patients (Figure 2, Table 1). Of note, for patients 10, 11, and 15 the multiple isolates obtained from each were identical, suggesting that the short incubation periods did not support extensive within-host diversification. However, isolates from patient 1 were represented by multiple genetic subtypes of ST191 (clades A and C), which were differentiated by 20 core genome SNPs (Figure 2). Of the four subtypes, three were identified in multiple patients, including subtype A in four patients, subtype B in seven patients, and subtype C in two patients, indicating their wide distribution among patients infected during the outbreak (Table 1). The short timescale between exposure to and isolation of the pathogen during the outbreak and the lack of person-to-person transmission for L. pneumophila [33,34] strongly suggest that the genetic subtypes of ST191 existed in the outbreak source prior to release and evolved from a recent progenitor within the water reservoir by a combination of gene mutation and recombination.Figure 2

Bottom Line: In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution.Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Legionnaires' disease is a severe form of pneumonia caused by the environmental bacterium Legionella pneumophila. Outbreaks commonly affect people with known risk factors, but the genetic and pathogenic complexity of L. pneumophila within an outbreak is not well understood. Here, we investigate the etiology of the major Legionnaires' disease outbreak that occurred in Edinburgh, UK, in 2012, by examining the evolutionary history, genome content, and virulence of L. pneumophila clinical isolates.

Results: Our high resolution genomic approach reveals that the outbreak was caused by multiple genetic subtypes of L. pneumophila, the majority of which had diversified from a single progenitor through mutation, recombination, and horizontal gene transfer within an environmental reservoir prior to release. In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution. Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.

Conclusions: Taken together, our study indicates previously cryptic levels of pathogen heterogeneity within a Legionnaires' disease outbreak, a discovery that impacts on source attribution for future outbreak investigations. Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

Show MeSH
Related in: MedlinePlus