Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

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1 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47 M. Mentasti, P. Cassier, S. David, C. Ginevra, L. Gomez-Valero, A. Underwood, B. Afshar, J. Etienne, Julian Parkhill, V. Chalker, C. Buchrieser, T. G. Harrison and S. Jarraud; ESCMID Study Group for Legionella Infections (ESGLI). ESGLI 2016, Amsterdam, The Netherlands (22 September 2016)

2 Tracklist 1 Background 2 Legionella pneumophila sg1 ST47 3 LPO_1073 real time PCR assay 4 Evolution of ST47 5 Conclusions 6 Acknowledgments 7 References 2 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

3 L. pneumophila SBT database 3 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

4 England & Wales Comparison of clinical and routine environmental isolates in England and Wales between 2000 and 2008: n sg1 Pontiac Clinical % 91.6% Environmental % 8.3% (Harrison et al., 2009) L. pneumophila sg1 Pontiac (mab 3/1 +ve) strains are the main cause of LD but are less frequently isolated from the environment. 4 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

5 Environmental isolates 20 ST1 15 ST79 82 different STs identified ST1 + ST79 = 34.1% % 10 5 ST47 0 NEW F05 F04 F22 F20 F02 F16 F12 F11 F F25 F24 F19 F F F03 F F17 F F F ST 5 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

6 Clinical isolates 20 ST different STs identified ST47 alone causes 22.8% of LD cases % 10 5 ST1 0 NEW F05 F04 F22 F20 F02 F16 F12 F11 F F3 F2 F serogroup ST 6 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

7 L. pneumophila sg1 ST47 Main cause of LD in Northern Europe: - England and Wales (Harrison et al., 2009) = 22.8% of LD cases; - France (Ginevra et al., 2008) = 11.0% of LD cases; - Belgium (Vekens et al., 2012) = 27.9% of LD cases; - The Netherlands (Euser et al., 2013) = 41.0% of LD cases. Only occasionally isolated from the environment (almost all ST47 LD cases remain unresolved!). In the Netherlands ST47 isolated from soil samples using amoeba co-culture (Shalk et al., 2014). Are we analysing the right environmental samples? Are we routinely using the right approach to isolate ST47?!!! Specific ST47 assay needed to improve investigation of ST47 LD cases!!! 7 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

8 ST47 target(s) identification Complete or draft genome sequences available for 32 different STs representative of L. pneumophila species diversity (Underwood et al., 2013). Three ST47 strains [Lorraine (LPO, NC ), H and H ] included in the study. Annotated genes from the complete or assembled genomes subjected to an all by all BLASTn analysis using a threshold of e <= A blast hit was considered to be significant if there was >=90% nucleotide identity over >=90% of the length of the sequence. Comparative analysis identified 64 possible ST47 specific targets. 8 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

9 ST47 target selection Primers designed to amplify detect 21/64 targets as: - present on the ST47 chromosome and not on the (150kb) plasmid - No hit against the NCBI database using BLASTn A collection of 95 (NCTC - ATCC - CIP) strains tested to evaluate the targets: - 16 L. pneumophila sg1 ST47; - 16 L. pneumophila sg1 non-st47; - 16 L. pneumophila serogroup 2-16 reference strains; - 45 non-pneumophila Legionella spp.; - 16 non-legionella strains. LPO_1073 resulted ST47 specific. Real-time PCR designed to amplify a 142bp fragment of LPO_ Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

10 LPO_1073 assay in silico evaluation 486 genomes published in other studies (n=423) or sequenced for this study (n=63) were used to determine the specificity of LPO_1073 assay. LPO_1073 resulted 100% conserved in all 129 available ST47 genomes. LPO_1073 was not detected in any of the 357 non-st47 strains (77 different STs) for which WGS data were available at the time of testing. LPO_1073 is a part of a of 16,266bp fragment inserted at a trna which comprises 12 genes annotated as phage-related genes. PFAM ( did not find any match between LPO_1073 translated sequence and known protein families, thus its function is unknown. 10 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

11 LPO_1073 assay in vitro evaluation (1) The specificity of the LPO_1073 real time assay was evaluated on 452 strains. All 110 L. pneumophila ST47 isolates gave a positive result, which is concordant with the in silico detection of LPO_1073 in all ST47 genomes. 26 out of 44 ST109 strains isolated in France (11), Belgium (1), Denmark (2), Germany (5) and the Netherlands (7) were detected by the assay. The remaining 18 ST109 strains isolated in England & Wales (11), the USA (5) and Germany (2), were not detected by the assay. ST109 WGS data not available, thus this finding had not been uncovered during the in silico evaluation. 11 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

12 LPO_1073 assay in vitro evaluation (2) Efficiency = 0.98 (SD = 0.06) Limit of Detection (95% confidence) = 34.3 copies/reaction Clinical samples: LPO_1073 concordant with previous culture + (n)sbt results obtained on 106 respiratory samples from LD patients. Environmental samples: A total of 3 samples from 2 sources that were culture positive for ST47 gave a strong positive LPO_1073 PCR result (ca genome copies in 5 µl of DNA extract). 12 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

13 ST47/ST109 phylogenetic structure (1) WGS was applied to decipher the positive LPO_1073 PCR results obtained with the 26 ST109 (DLV) isolates: - 9 France ST109 isolates (LPO_1073 positive); - 8 England & Wales ST109 isolates (LPO_1073 negative); ST47 isolates (LPO_1073 positive); - 6 ST74 isolates (LPO_1073 negative); - 4 ST82 isolates (LPO_1073 negative). The in silico analysis detected LPO_1073 in the 9 France ST109 genomes but not in 8 England & Wales ST109 genomes. Sequence reads were mapped to the Lorraine ST47 genome and phylogenetic analysis was performed before and after the removal of recombined regions in the alignment. 13 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

14 Phylogenetic analysis (1) ST47 isolates form a highly clonal, monophyletic clade with a maximum of just 33 SNPs between any pair. The 9 France ST109 isolates cluster most closely to the ST47 clade (3116 to 3175 SNPs observed between any ST47 and any France ST109 isolate). The 8 England & Wales ST109 isolates cluster more distantly from the ST47 clade (5497 to 7737 SNPs between isolates from the two groups). ST74 and ST82 isolates are even more divergent (a maximum of 12,415 and 22,911 SNPs from ST47 isolates, respectively). Single acquisition of the fragment carrying LPO_1073 by a common ancestor of ST47 and France ST Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

15 Phylogenetic analysis (2) Recombined regions were searched and removed using Gubbins. 98.9% of SNPs in the original alignment had been imported via recombination events. Dramatic reduction of branch lengths!!! The different STs now differ by only a small number of de novo mutations. France ST109 isolates now cluster very closely with ST47 isolates, with just 4 to 29 SNPs between any pair from the two groups. Seven France ST109 isolates now cluster within the ST47 lineage itself while another two form an outgroup to the ST47 lineage. However, this could be artefact due to the very low number of SNPs that differentiates them. 15 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

16 Evolutionary analysis (1) Almost all the SNP differences observed were imported via recombination. Hypothesis: since ST47 causes LD more frequently than France ST109, the recombined regions imported into ST47 since its divergence with France ST109 may be very important for ST47 high disease prevalence and/or virulence. Gubbins detected 2 regions imported into ST47 via homologous recombination that differentiate ST47 from France ST109. The first is a 170kb region containing 3013 SNPs (i.e. 98.2% nucleotide similarity to France ST109). The second is a 9kb region containing 148 SNPs (i.e. 98.4% nucleotide similarity to France ST109). When the two regions were used as queries in a BLASTn search against L. pneumophila genomes, both were found to be extremely similar to ST62 genomes. ST62 is a well documented cause of LD. 16 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

17 Evolutionary analysis (2) 7 SNPs were found in the first region of ~170kb between the Lorraine (ST47) strain and a representative ST62 strain (H ), while 0 SNPs were found in the ~9kb region. Strong evidence that these recombined regions were imported into an ST47 ancestor from an ST62 or ST62-like strain. Recombination events that occurred earlier in the tree (i.e. prior to the divergence of ST47 and France ST109) also indicate that several adjacent regions have been imported from the ST62 lineage on multiple occasions. ST47 shares ~13.5% of the chromosome with ST62! Large region of high similarity between the five major disease-associated STs described by David et al. (2016). These recombination events also explain why ST47 shares 3 identical SBT alleles (pile, mip and neua) with ST62 strains, while another SBT allele (proa) differs by only one SNP. 17 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

18 Conclusions WGS used for the first time on a large collection of sequenced L. pneumophila genomes to identify a molecular target for a specific clade of L. pneumophila sg1 strains. A sensitive and reliable real time PCR assay developed to preliminarily detect and identify ST47 & France ST109 directly from primary samples. LPO_1073 PCR could be used as a screening tool followed by culture of positive samples (including amoebae co-culture) to increase recovery in so far unexplored environmental niches. Finally, with WGS becoming more and more accessible, the genome of clinical strains isolated during outbreaks could be quickly sequenced and a specific molecular assays designed to screen environmental samples and aid the identification of the possible source. 18 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

19 Acknowledgments Natalia Kozak-Muiznieks (USA), Soren Uldum (Denmark), Carla Duncan (Canada), Linda van de Nes-Reijnen (The Netherlands), Alex Ensminger (Canada), Fedoua Echahidi (Belgium) and Christian Lueck (Germany) for providing strains and/or DNA and/or sequences. Work in BBI is financed by the Institut Pasteur, the Institut Carnot-Pasteur MI, the French Region Ile de France (DIM Malinf), the grant n ANR-10-LABX-62-IBEID and the Fondation pour la Recherche Médicale (FRM) grant N DEQ Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

20 References Euser SM, Bruin JP, Brandsema P, Reijnen L, Boers SA, Den Boer JW. Legionella prevention in the Netherlands: an evaluation using genotype distribution. Eur J Clin Microbiol Infect Dis. 2013; 32(8): Ginevra C, Forey F, Campese C, Reyrolle M, Che D, Etienne J, et al. Lorraine strain of Legionella pneumophila serogroup 1, France. Emerg Infect Dis. 2008; 14(4): Harrison TG, Afshar B, Doshi N, Fry NK, Lee JV. Distribution of Legionella pneumophila serogroups, monoclonal antibody subgroups and DNA sequence types in recent clinical and environmental isolates from England and Wales ( ). Eur J Clin Microbiol Infect Dis. 2009; 28(7): Schalk JA, Euser SM, van Heijnsbergen E, Bruin JP, den Boer JW, de Roda Husman AM. Soil as a source of Legionella pneumophila sequence type 47. Int J Infect Dis. 2014; 27:18-9. Underwood AP, Jones G, Mentasti M, Fry NK, Harrison TG. Comparison of the Legionella pneumophila population structure as determined by sequence-based typing and whole genome sequencing. BMC Microbiol. 2013; 13:302. Vekens E, Soetens O, De Mendonca R, Echahidi F, Roisin S, Deplano A, et al. Sequence-based typing of Legionella pneumophila serogroup 1 clinical isolates from Belgium between 2000 and Euro Surveill. 2012; 17(43): Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47

21 !!! Many thanks for your attention!!! 21 Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 ST47