Legionella Infections

Molecular detection and typing of Legionella infections

Sophie JARRAUD

ESCMID eLibrary © by author Disclosure

Research Grants, consulting:

Diagenode, IDEEX, bioMerieux, Orgentec, Alere

ESCMID eLibrary © by author Legionella Legionnaires’ disease (LD) infections Pontiac fever = ü atypical pneumonia (eg. legionellosis ü a mild, flu-like disease extrapulmonary findings) ü without pneumonia ü severe pneumonia ü high attack rate ü Mortality: 10-30% ü short incubation period ü rapid recoveries Extrapulmonary forms

ü with or without pneumonia ü especially due to L. non pneumophila ESCMIDü endocarditis, eLibrary arthritis … © by author Legionella associated to LD genus Legionella ~ 25 species: at least once isolated from humans

L. pneumophila ~ 91% L. longbeachae ~ 5% cases worldwide ~ 60 L. micdadei ~ 2% species (with isolates available) Other species ~ 2% (Yu et al., J. infect. Dis. 2002) Legionella longbeachae : ~ 30% - 50% of the cases in Australia and New Zealand

L. pneumophila is clearly predominant in human infection followed ESCMIDby L.eLibrary longbeachae © by author Legionella associated to LD

Legionella pneumophila : ~ 91% of the cases worldwide (Yu et al., J. infect. Dis. 186:127-128)

16 L. pneumophila sg1: ~ 88,6% of cases serogroups

~ 30% of isolates from environment

Higher virulence or better niche adaptation of ESCMIDLegionella eLibrary pneumophila Sg 1? © by author What is the use of Legionella PCR in Europe?

Beauté J. et al. Legionnaires’ disease in Europe, 2011 to 2015. Eurosurveillance 2017

33,809 laboratory tests 29 reporting countries PCR, 6,8% other tests culture, (serology), 10,8% 1,1% Proportion of cases diagnosed by PCR increased 4.3% (2011) to 10.5% (2015)

urinary antigen, ESCMID eLibrary78,2% © by author What are major advantages of Legionella PCR ?

1- main advantage: diagnosis of all Legionella spp. infections

– largely unknown and suspected to be under-diagnosed – Legionella sp.: 16S rRNA, 5S rRNA, intergenic spacer region 23S-5S, • Species identification available after sequencing – L. pneumophila: mip gene (or other)

ü L. non-pneumophila associated to immunocompromised patients ü Higher mortality due to these patients ESCMID eLibrary © by author 2- qPCR as a pronostic marker?

q In respiratory samples Maurin et al., CMI, 2010 (n = 70); Murdoch et al., CID, 2013 (n = 114) • Correlation of bacterial load on admission with • Pneumonia severity index (Fine class or CURB-65) • Need for hospitalization in ICU • Duration of hospitalization • Mortality

q limitation: standardization sampling procedure

q Sputum and tracheal aspirate provide better results than Bronchial aspirate samples • higher positive rates ESCMID• higher bacterial loads eLibrary © by author 2- qPCR as a pronostic marker?

q In blood samples

Lindsay et al., 1994; Murdoch et al., 1996; Murdoch et al., 1999; Murdoch et al., 2000; Matsiota-Bernard et al., 2000; Diederen et al., 2007; Van de Veerdonk et al. 2009; Mentasti et al., 2012 • ESCMIDCorrelation between PCR on serum eLibrary / LD severity not clear-cut © by author 3- Molecular method for detecting antibiotic resistance

• resistance to antibiotics seems not a major problem for LD – Only 2 descriptions of resistance to Quinolone ! • 1 isolate from patient (Bruin et al. JAC 2014) • 1 description by targeted NGS (Shadoud et al. EBIOM 2015)

– No description (from patient / environment) for macrolides and Rifampicin

• Isolates available for only 12% of cases (diagnosed in Europe)

• Development of molecular methods to confirm these data and to perform surveillance ESCMID eLibrary © by author 3- Molecular method for detecting antibiotic resistance

• Molecular mechanisms associated to resistance determined on in vitro selected resistant mutants (Nielsen et al., 2000; Almahmoud et al., 2009; Descours et al., 2017)

PCR and real time PCR assay Digital PCR Targeted sanger sequencing targeting mutations NGS method

Resistant Quantification FQ resistance WT 1:1,000 resistant/ susceptible allele ratios

Almahmoud et al., 2009 Hennebique et al., 2017 Shadoud et al. 2015 ESCMIDShadoud et al. 2015 eLibrary © by author Targeted Next Generation Sequencing (NGS)

ü ability to detect minority subpopulations ü demonstration of in vivo selection of FQ-resistant mutants of Legionella for 2 patients during hospitalisation Shadoud et al. EBIOM (2015)

Day of sampling % of gyrA83 mutant (D0= diagnosis) alleles

Patient #2 D0 2.9 % D4 94 % Patient #4 D0 1.05 % D3 75 % D5 95 % % ESCMIDof mutated alleles in respiratory sampleseLibrary from control patients: <0.5% © by author 4- Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections

Why does Legionella a good candidate as target ?

ü Severe pneumonia: rapid diagnosis ü Several mimics of LD: rapid differential diagnosis ü Potential co-infection: influenza virus / others ? ü Urinary antigen tests detect only Lp1

ESCMID eLibrary © by author ePlex® BIOFIRE® FILMARRAY® Respiratory Pathogen Pneumonia Panel (GenMark Diagnostics) (bioMéreux)

Upper respiratory infection Lower respiratory infection

Panels available for Legionella pneumophila DiaCORE® Unyvero® HPN Respiratory Panel (Curetis) (STAT Dx) Hospitalized pneumonia ESCMID eLibrary © by author ePlex® nasopharyngeal swabs not adapted to LD Respiratory Pathogen (GenMark Diagnostics)

Upper respiratory infection CE-IVD cleared for nasopharyngeal swabs only

R. H. T. Nijhuis, J Clin Microbiol. 2017 Analysed 6 LD from sputum = positive result

DiaCORE® Respiratory Panel (STAT Dx)

nasopharyngeal swabs ESCMIDNo publication eLibrary © by author Authorization seeked from the FDA BIOFIRE® FILMARRAY® Pneumonia Panel ü Sputum - endotracheal aspirate (bioMéreux) ü Bronchoalveolar Lavage ü No publication Lower respiratory infection ü Poster at ECCMID: only few LD tested

Unyvero® HPN ü No publication (Curetis) ü Poster at ECCMID: only few LD tested Hospitalized pneumonia ü Incidence of LD is low ü We need also evaluation from National Centers ESCMIDof Legionella eLibrary © by author 5- 16S rRNA targeted metagenomic on clinical samples

Which applications for Legionella infections?

• Diversity of bacteria in clinical samples from LD – What are the dynamics of the lung microbiome and the pathogen during Legionella infection and antibiotic treatment? – Are there other bacteria needed to treat?

• Legionella species identification – Are there several Legionella species in the clinical sample? ESCMIDLung microbiome during LDeLibrary - only very few published studies © by author What are the dynamics of the lung microbiome and the pathogen during Legionella infection and antibiotic treatment?

Ana Elena PEREZ COBAS Carmen Buchrieser’s team

Christophe Christophe Ginevra Rusniok ESCMID eLibrary © by author Persistent Legionella infection during hospitalisation

Thoracic CT scan: chest X-ray a voluminous lung abscess Resection of abscess

rapid clinical improvement D4 D5 D14 D24 D33 D42 D54 D62 D72

Erythromycin + Erythromycin + nitroimidazole Levofloxacin levofloxacin + levoflo + imidazole + Rifampicin (3 months)

ESCMIDBAL: Legionella positive culture eLibrary © by author METHODS Illumina technology PCR 16S rRNA V3-V4 ITS1

Operational taxonomic units

Bioinformatics algorythms

ESCMID eLibraryAdapted from Hong et al , 2016 Clin. Microb. Rev. © by author RESULTS 16S rRNA sequencing Antibiotics 100 Actinobacteria Actinomyces Actinobacteria Atopobium Legionella Actinobacteria Corynebacterium Bacteroidetes Alloprevotella Bacteroidetes Prevotella 80 Firmicutes Abiotrophia Firmicutes Enterococcus Firmicutes Gemella Firmicutes Granulicatella Firmicutes Oribacterium Firmicutes Peptostreptococcus 60 Firmicutes Shuttleworthia Firmicutes Staphylococcus Firmicutes Streptococcus Firmicutes Veillonella Firmicutes uc_Lachnospiraceae Fusobacteria Fusobacterium 40 Proteobacteria Haemophilus Proteobacteria Legionella Tenericutes Mycoplasma Others

20 Very high abundance of Legionella in the microbiome during infection (75%)

0 Antibiotic therapy changes the diversity of the microbiome due to resistant species day 5 BAL 5 day day 24 BAL 24 day BAL 33 day BAL42 day day 14 BAL 14 day

day 4 sputum 4 day (Prevotella, Alloprevotella, Fusobacterium) ESCMID abscess 42 day eLibrary © by author RESULTS Relative abondance (%)

ESCMID eLibrary100 60 80 20 40 0

day 4 sputum Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Bacteroidetes Bacteroidetes Actinobacteria Actinobacteria Others Proteobacteria Firmicutes Firmicutes sample sample differentHighly of BAL composition microbiome sputum and Legionella Peptostreptococcus Granulicatella Gemella Abiotrophia Veillonella Streptococcus Prevotella Alloprevotella Atopobium Actinomyces Legionella

High-abundance > 2% taxa SputumBAL versus 16S 16S Legionella BAL Legionella Gemella Prevotella Streptococcus Sputum rRNA (13%) (13%)

sequencing (13%) (13%) (99%) (3%) (43%) (43%)

RESULTS

Relative abondance (%) 100 60 80 40 20 ESCMID0 eLibrary

©day by 5 BAL author

day 4 sputum Others uc_Proteobacteria Proteobacteria uc_Pasteurellaceae Proteobacteria uc_Gammaproteobacteria Proteobacteria uc_Chromatiales Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Fusobacteria Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes Firmicutes uc_Cyanobacteria Cyanobacteria uc_Thermoprotei Crenarchaeota Bacteroidetes Bacteroidetes Bacteroidetes uc_Actinomycetales Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Veillonella Streptococcus Staphylococcus Peptostreptococcus Granulicatella Gemella Exiguobacterium Enterococcus Atopostipes Abiotrophia Fusobacterium Flavobacterium Alloprevotella Prevotella Propionibacterium Microbacterium Corynebacterium Atopobium Actinomyces Rheinheimera Pseudomonas Neisseria Haemophilus Brevundimonas Aeromonas Acinetobacter i species of low-abundant Detection

n n the BAL samples Low-abundance < 2% taxa SputumBAL versus 16S 16S rRNA

sequencing RESULTS 16S rRNA sequencing 100 Bacteroidetes Alloprevotella Bacteroidetes Prevotella Firmicutes Oribacterium Firmicutes Shuttleworthia Firmicutes Staphylococcus Firmicutes uc_Lachnospiraceae 80 Fusobacteria Fusobacterium Proteobacteria Legionella Tenericutes Mycoplasma Others Abscess versus BAL

60 High-abundance taxa > 2%

Fusobacterium 40 Abscess Higher presence of pathogenic bacteria Legionella (38%) 20 Legionella Fusobacterium (15%)

0 Fusobacterium nucleatum (abscess culture) day 42 BAL 42 day

ESCMIDabscess42 day eLibrary © by author Lung microbiome composition during LD

• BAL samples >> sputa samples: real lung microbiome composition during Legionella infection

• Very high abundance of Legionella in the microbiome during infection

• Several pathogens are present in the lung abscess

• After antibiotic therapy we observed a change on the biodiversity of the microbiome due to resistant species

ESCMID eLibrary © by author Persistent Legionella infection or LD complications Are there other bacteria needed to treat?

Rapid bacterial composition analysis by real-time Nanopore sequencing of the same abscess

16S barcoding kit Nanoporetech Ready-to-use kit amplification of the full-length of the16S rRNA genes (~1400bp) Barcoted primers Multiplexing of 12 samples

Sequencing on an Oxford Nanopore TechnologiesESCMID MinION sequencer eLibrary © by author Workflow (1)

Clinical sample extraction

PCR products Purification Quantification, dilution & pool

Library preparation ESCMID eLibrary © by author Workflow (2)

Base-calling, demultiplexing and BlastN search using 2 tools

Blast fastQ against real-time BlastN search (Epi2ME) RefSeq Database (One Codex)

ESCMID eLibrary © by author RESULTS Real-time BlastN search (Epi2ME) first results <30 min after loading

ESCMID eLibrary © by author Real-time BlastN search (Epi2ME) Illumina technology

100 Bacteroidetes Alloprevotella Bacteroidetes Prevotella Firmicutes Oribacterium Prevotella Firmicutes Shuttleworthia Firmicutes Staphylococcus Firmicutes uc_Lachnospiraceae 80 Fusobacteria Fusobacterium Proteobacteria Legionella Tenericutes Mycoplasma Oribacterium Others

Staphylococcus60

Fusobacterium 40

Legionella20

0 day 42 BAL 42 day ESCMID eLibraryabscess42 day © by author Real-time BlastN search (Epi2ME) Illumina technology

Staphylococcus60

Fusobacterium 40

Legionella20

0 Similar results obtained by the two technology day 42 BAL 42 day ESCMID eLibraryabscess42 day © by author Real-time BlastN search (Epi2ME) Illumina technology

Staphylococcus60

Fusobacterium 40

Legionella20

MinION technology could be interesting0 for a rapid bacterial composition analysis in a context of LD complications day 42 BAL 42 day ESCMID eLibraryabscess42 day © by author Example 2 – using MinION technology for rapid Legionella species identification in respiratory samples

CONTEXT • At the same time - in the same area: two Legionnaires’ disease due to Legionella non-pneumophila – by using a specific Legionella sp. PCR – Sending of samples to national reference center for species identification: sequencing of intergenic spacer region 23S-5S L. gratiana: never isolated from patients

è same source of contamination for the 2 patients?

Interest of MinION technology: rapid result for a rapid epidemiological investigation? ESCMID eLibrary © by author RESULTS 64,336 reads classified

ESCMID eLibrary © by author RefSeq Database (One Codex)

ESCMIDLegionella gratiana eLibrary for the 2 samples © by author Example 3 - Are there several Legionella species in the clinical sample?

• Legionella bozemanii arthritis • 5 days after a fall on the wet floor à superficial wound on the middle left finger • Surgery at Day 20: 4 joint fluids – Standard culture: negative – PCR - Sequencing 16s rRNA Positive for L. bozemanii – Confirmation by 23S-5S ribosomal intergenic sequencing ESCMID eLibrary © by author Specific culture: BCYE, BMPA and MWY 1 sample/4 positive after 9 days on BCYE agar

• 5 isolates identified as L. bozemanii • Various sizes and colour strains è Infection due to multiple Legionella ? • InESCMID support to direct contamination eLibrary after a fall on a wet floor © by author Blast fastQ against RefSeq Database (One Codex)

490 reads only one species: Legionella bozemanii

ESCMID eLibrary © by author Typing of Legionella for epidemiological investigations

ESCMID eLibrary © by author L. pneumophila international SBT database

ESCMID eLibrary © by author 5 STs represent ≈ 40% of all clinical isolates

20 Clinical 15 Environmental

Percentage of databaseSBT 5 isolates submitted to to isolatessubmitted 0 ST1 ST23 ST37 ST47 ST62

Except ST1 these ST are rarely isolated from the environmental ESCMIDsources eLibrary investigated © by author Usefulness of PCR for epidemiological investigations

Sg1-specific PCR Merault N. et al. 2011 Mentasti M. et al. 2015

Nested-SBT directly on clinical samples

Ginevra et al. 2009 ; Coscola et al. 2009 ; Mentasti et al. 2012 ESCMID eLibrary © by author Usefulness of PCR for epidemiological investigations

Sg1-specific PCR

Nested-SBT directly on clinical samples

Specific PCR of clones

ESCMIDST47-specific eLibrary PCR Mentasti et al. CMI 2017 © by author ST1-specific PCR

Ø Worldwide distribution Ø Frequent in environmental samples Cazalet Nat Genet 2004, Ginevra CID 2009, Cassier P. New Microbes New Ø High mortality: 30% Infect. 2014, David S. et al. CID 2017, Ø Associated to hospital-acquired infections Ø Associated to immuno-compromised patients

PCR can be useful for screening of Lp1 isolates for further WGS analysis ESCMID eLibrary © by author Study Workflow Minimum spanning trees of L. pneumophila isolates Categorical clustering based on ST was performed using Bionumerics 7.1 software

Whole genome sequencing of Lp1 477 Number of different Alleles PCR ST1 results

isolates 922 1 922 - 2 1380 131 genomes, 49 ST1 3 + 1163 4 704 224 1 PCR assay 704 45 > 4 232 94 Specificity to ST1 Complex clonal 117 Identification of 12 putative ST1 , 65, 65 , 1904, 1904 specific targets 624 1358 1237 580 701 2116 1327 , 1284, 1284 435 1570 743 455 995 18 , 266, 266 23 20 Validation on 143 culture 22 506 702 22 506 1085 763 1292 Primers and taqman probes design 1292 891 positive respiratory samples 96 75 , 575, 575 1901 1348 1348 760 - 20 ST1 569 708 256 708 542

In silico validation on international 1430 239 - 123 non-ST1 è 1 1430 407 407

whole genomes data sets 369 1386 680 1488 1419 1488 169 579 genomes, 113 ST1 discordance: suspicion of 81 ST1 clonal 646 778 6 540 778 630 540 complex 751338 177 2046 coinfection? 87 152 1240 1022 , 534, 534 10 709 1797 1691 Primers and probes design 848 474 1691

optimisation 1822 779 7 , 1, 11019 390 7 targets remaining 1113 476 35 2130 759 759 37 700 1946 211181

Validation on water samples have to be 1102, 9 1285 1285 40 PCR specificities on PCR sensitivities on 107 performed 664553 L. pneumophila DNA serial dilution of 1455 128

188 Lp, 97 ST1 and 59 L. pneumophila Paris 59 733

313 related to STs CIP107629 DNA 1044 ESCMID eLibrary 948 © by author Usefulness of PCR for epidemiological investigations

Sg1-specific PCR

Nested-SBT directly on clinical samples

specific PCR of clones

Identification of subspecies of ESCMIDL. pneumophila eLibrary © by author Distribution of L. pneumophila subspecies in infections ?

A recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters

Description of the fourth L. pneumophila subspecies

L. pneumophila subsp. pneumophila L. pneumophila subsp. fraseri L. pneumophila subsp. pascullei L. pneumophila subsp. raphaeli This subspecies consists of 15 STs

ESCMID eLibraryKozak-Muiznieks NA et al. Infect Genet Evol . 2018 © by author Lp subsp. fraseri Phylogeny of L. pneumophila Lp subsp. raphaeli Lp subsp. pascullei each subspecies forms a distinct phylogenetic clade within the L. pneumophila species

Lp subsp. pneumophila

ü Large number of Lp subsp. raphaeli Lp subsp. raphaeli strains compared to subsp. fraseri (ST259, ST701, and pascullei ST1904, ST2131) ü Belonging to 4 STs ü 2 news: ST701 and 1904 Lp subsp. fraseri Lp subsp. pascullei

Lp subsp. pneumophila

267 genome ESCMID assemblies from French ReferenceeLibrary Center of Legionella © by author Clinical incidence of L. pneumophila subsp. raphaeli ?

Data base of Legionella clinical isolates from French Reference Center Percentage of STs corresponding to Lp subspecies raphaeli

9,0% 31 8,0% 7,0% 26 18 22 ST 701 6,0% 18 19 16 ST 259 5,0% 10 ST 1904 4,0% ST 2131 3,0% 7 2,0% 1,0% 3 0 0,0% 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 LpESCMID subsp. raphaeli represent abouteLibrary 5% of all French clinical isolates © by author Ability of established typing methods to separate Lp strains into subspecies ?

ü Serogroup determination No ü mip typing ü 16S RNA sequences: 2 groups ü subsp. fraseri, raphaeli and pascullei have identical sequences ü different from subsp pneumophila gyrB gene: clear separation of all four subspecies

Kozak-Muiznieks ESCMID NA et al. 2018 PhylogeneticeLibrary tree (gyrB sequences of Lp subspecies) © by author Typing of Legionella for epidemiological investigations

Lack discriminatory power of SBT: sometimes unable to distinguish between the strains isolated from different environmental sources

• Mainly due to extensive recombination events and horizontal gene transfer

• mutation rate very low – evaluated for 2 STs: 0,49 and 0.71 SNPs/genome/year

David S et al. PLoS Genet. 2017 ; David et al. Genome Research 2016 ; Gomez-Valero L. BMC Genomics. 2011 ; CazaletESCMID C. Genome Res. 2008 eLibrary © by author SNP-based method

Sequence reads

Sequence reads mapping on reference genome Reads from Strain X Detection of Single Nucleotide Polymorphisms Reference genome (SNP) To be performed for the XX clinical isolates ESCMIDRaxML tree eLibrary © by author core genome MLST (cgMLST)

Sequence reads

De novo assembly Blast

Blast against a predefined cgMLST scheme/database

2016

Isolates tested • 106 isolates from ESGLI Panel Typing • 229 isolates (195 from 5 STs) ESCMID eLibrary © by author Typability

ü n = 335 isolates Method Typability rMLST (53 ribosomal) 0.899 cgMLST (50 genes) 0.988 ü Typability cgMLST (100 genes) 0.988 decreases as cgMLST (500 genes) 0.973 the number of cgMLST (1455 genes) 0.916 genes increases cgMLST (1521 genes) 0.379 SNP-based 0.988

ü using 79 epidemiologically unrelated isolates from the typing panel

Method Index of discrimination SBT 0.940 SBT + Mab subgrouping 0.968 rMLST (53 ribosomal) 0.972 ≥ 0.99 cgMLST (50 genes) 0.990 cgMLST (100 genes) 0.991 cgMLST (500 genes) 0.997 cgMLST (1455 genes) 0.998 cgMLST (1521 genes) 0.999 SNP-based 0.999 Most discriminatory

50-gene cgMLST achieves high discrimination (D=0.990) whilst maintaining good epidemiological concordance

ESCMID eLibrary © by author An international Working Group on NGS for Legionella • Group members: – Jacob Moran-Gilad, Jerusalem, Israel (chair, on behalf of ESGLI) – Kathy Bernard, Winnipeg, Canada – Vicki Chalker, London, UK – Alex Ensminger, Toronto, Canada In few months, proposals of a – Sophie Jarraud, Lyon, France standardized 50-genes cgMLST – Natalia Kozak-Muiznieks, Atlanta, USA – Christian Lück, Dresden, Germany scheme / database – Brian Raphael, Atlanta, USA – Rodney Ratcliff, Adelaide, Australia Essential for international surveillance – Søren Uldum, Copenhagen, Denmark of LD

• Expert advisors: – Anthony Underwood, London, UK - bioinformatics expert – Sophia David, Cambridge, UK – bioinformatics expert ESCMID– João André Carriço, Lisboa, Portugal eLibrary - bioinformatics expert © by author WGS has been applied to several LD outbreaks

and more …

Legionnaires’ disease in immunocompromised patients: beware of toilets!

ESCMID eLibrary © by author Two nosocomial LD in immunocompromised patients hospitalised in the same room (haematology unit)

5 months

16/12/2016 24/12/2016 08/01/2017 04/05/2017 28/05/2017 31/05/2017 01/06/2017

Patient 1 Patient 2 Environmental investigations

water from shower & bathroom sink was filtered Lp1 (+) BAL Lp1 (+) BAL

• 17 ST1 Lp1 strains available isolated from: – Toilet bowl of the patients: N=4 (1100 UFC/L) – Toilet bowl of the closest room: N=2 – 2 other water network samples in the unit: N=3 (10 and 20 CFU/L) – different area from hospital, outside the unit: N=8

• Sequencing: Nextera XT PE 2x150bp • Genome analysis: Nullarbor pipeline • Phylogenetic tree containing other unrelated ST1 strains (from other LD investigations) ESCMID eLibrary © by author 5 SNPs

Toilet bowl – adjacent room Patient 1

Patient 2 4 strains from toilet bowl (room n°1)

RAxML onESCMID core-full-aln (Including invariant sites) eLibrary after recombination sites removal by Gubbins © by author 5 SNPs

Toilet bowl – adjacent room Patient 1

Patient 2 4 strains from toilet bowl (room n°1)

• All isolates clustered together and share the same most recent common ancestor • 0 SNP: patient 2 and 4 Lp1 from the toilet bowl of its own room èSource of contamination of this patient RAxML onESCMID core-full-aln (Including invariant sites) eLibrary after recombination sites removal by Gubbins © by author Conclusion • The detection of Legionella non pneumophila by PCR should be favored in addition to L. pneumophila detection

• The use of rapid syndromic testing could increase the number of diagnosed LD

• The rapid bacterial composition analysis of clinical sample by MinION (or other technology) during Legionella infection could be interesting

• The shotgun metagenomic analysis of respiratory samples is the next step

• An international standardized 50-genes cgMLST scheme will be soon ESCMIDproposed – essential for epidemiological eLibrary investigation of LD © by author ESCMID eLibrary © by author Acknowledgments

Carmen BUCHRIESER Ana Elena PEREZ COBAS Christophe RUSNIOK Christophe GINEVRA Joëlle CHASTAING Ghislaine DESCOURS Jérémy REBOULET Laetitia BERAUD Marielle SIFFERT NESA N Anne Gaëlle RANC Noémie FESSY TANKOVIC J Gérard LINA Isabelle ROYET BARBUT F Florence ADER Corinne FAUCHON Nathalie JACOTIN Pierre Alain BILLY Patricia MARTIN-SIMOES

Legionella pathogenesis team, International Center of Research in ESCMIDInfectiology eLibraryChristine CAMPESE © by author