OBJECTIVES: Legionella pneumophila is the leading cause of Legionnaires' disease, a severe form of pneumonia acquired from environmental sources. Investigations of both sporadic cases and outbreaks rely mostly on analysis of a single to a few colony pick(s) isolated from each patient. However, because of the lack of data describing diversity within single patients, the optimal number of picks is unknown. Here, we investigated diversity within individual patients using sequence-based typing (SBT) and whole-genome sequencing (WGS). METHODS: Ten isolates of L. pneumophila were obtained from each of ten epidemiologically unrelated patients. SBT and WGS were undertaken, and single-nucleotide polymorphisms (SNPs) were identified between isolates from the same patient. RESULTS: The same sequence type (ST) was obtained for each set of ten isolates. Using genomic analysis, zero SNPs were identified between isolates from seven patients, a maximum of one SNP was found between isolates from two patients, and a maximum of two SNPs was found amongst isolates from one patient. Assuming that the full within-host diversity has been captured with ten isolates, statistical analyses showed that, on average, analysis of one isolate would yield a 70% chance of capturing all observed genotypes, and seven isolates would yield a 90% chance. CONCLUSIONS: SBT and WGS analyses of multiple colony picks obtained from ten patients showed no, or very low, within-host genomic diversity in L. pneumophila, suggesting that analysis of one colony pick per patient will often be sufficient to obtain reliable typing data to aid investigation of cases of Legionnaires' disease

Chalker, VJ

David, S

Mentasti, M

Parkhill, J

English

Apollo (Cambridge)

lable at ScienceDirectClinical Microbiology and Infection 24 (2018) 1020.e1e1020.e4Contents lists avaiClinical Microbiology and Infectionjournal homepage: www.cl in icalmicrobiologyandinfect ion.comResearch noteLow genomic diversity of Legionella pneumophila within clinicalspecimensS. David 1, *, M. Mentasti 2, J. Parkhill 3, V.J. Chalker 21) Centre for Genomic Pathogen Surveillance, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom2) Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England, Colindale, NW9 5EQ, United Kingdom3) Pathogen Genomics, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdoma r t i c l e i n f oArticle history:Received 5 January 2018Received in revised form22 February 2018Accepted 3 March 2018Available online 13 March 2018Editor: G. LinaKeywords:Genomic diversityLegionella pneumophilaLegionellosisLegionnaires' diseaseSBTWGS* Corresponding author. S. David, Centre for GenWellcome Genome Campus, Hinxton, Cambridge, CB1E-mail address: sd12@sanger.ac.uk (S. David).https://doi.org/10.1016/j.cmi.2018.03.0041198-743X/© 2018 The Author(s). Published by Elseviethe CC BY license (http://creativecommons.org/licensa b s t r a c tObjectives: Legionella pneumophila is the leading cause of Legionnaires' disease, a severe form of pneu-monia acquired from environmental sources. Investigations of both sporadic cases and outbreaks relymostly on analysis of a single to a few colony pick(s) isolated from each patient. However, because of thelack of data describing diversity within single patients, the optimal number of picks is unknown. Here,we investigated diversity within individual patients using sequence-based typing (SBT) and whole-genome sequencing (WGS).Methods: Ten isolates of L. pneumophila were obtained from each of ten epidemiologically unrelatedpatients. SBT and WGS were undertaken, and single-nucleotide polymorphisms (SNPs) were identiﬁedbetween isolates from the same patient.Results: The same sequence type (ST) was obtained for each set of ten isolates. Using genomic analysis,zero SNPs were identiﬁed between isolates from seven patients, a maximum of one SNP was foundbetween isolates from two patients, and a maximum of two SNPs was found amongst isolates from onepatient. Assuming that the full within-host diversity has been captured with ten isolates, statisticalanalyses showed that, on average, analysis of one isolate would yield a 70% chance of capturing allobserved genotypes, and seven isolates would yield a 90% chance.Conclusions: SBT and WGS analyses of multiple colony picks obtained from ten patients showed no, orvery low, within-host genomic diversity in L. pneumophila, suggesting that analysis of one colony pick perpatient will often be sufﬁcient to obtain reliable typing data to aid investigation of cases of Legionnaires'disease. S. David, Clin Microbiol Infect 2018;24:1020© 2018 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiologyand Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).IntroductionLegionella pneumophila is a Gram-negative bacterium found infresh-water and soil environments [1]. Human infection withL. pneumophila can cause legionellosis, which ranges from a mild,ﬂu-like illness (Pontiac fever) to a severe and potentially fatalpneumonia (Legionnaires' disease). The usual route of infection isvia inhalation of aerosols from a contaminated environmentalsource [2]. Commonly implicated sources include cooling towers,spa pools, decorative fountains, and water systems of largebuildings.omic Pathogen Surveillance,0 1SA, United Kingdom.r Ltd on behalf of European Societyes/by/4.0/).When Legionnaires' disease cases occur, clinical isolates areusually characterized together with epidemiologically linked envi-ronmental isolates to help determine the source of the infection. Todate, most clinical microbiological laboratories have relied on ana-lysing a single clinical isolate, or a small number of clinical isolates,from each patient. However, existence of within-host diversity ofL. pneumophila, which has been poorly studied, would have impor-tant implications for the interpretation of molecular typing data.Here, we used sequence-based typing (SBT) [3,4] together withwhole-genome sequencing (WGS) to investigate the diversityamongst multiple colony picks recovered from individuals.MethodsTen colony picks were obtained [5] from single sputumsamples of ten epidemiologically unrelated patients with sporadicof Clinical Microbiology and Infectious Diseases. This is an open access article underPs)identiﬁedamongsttenisolatesrecoveredfromeachoftenLegionnaires'diseasepatientsonEpidemiologicalinformationSTReferencegenome(andsizeinbp)Lengthofreferencegenomemapped(bp)Range,meanandmediannumberofSNPsbetweenpairsSNPlocationsandgeneAccessionnumbersNosocomialcase42EUL120(3,430,562)[6]3,376,590e3,378,6100SNPsbetweenallERR1608296eERR1608305Travel-associated42EUL120(3,430,562)[6]3,391,462e3,393,086Range0e2,mean0.4,median0470,661(puuB),2,941,541(hemC)ERR1608306eERR1608315y- hoea,Travel-associated(CaribbeanandSpain)42EUL120(3,430,562)[6]3,296,732e3,298,9090SNPsbetweenallERR2216341eERR2216350Travel-associated(Italy)23EUL28(3,509,586)[6]3,369,571e3,371,1480SNPsbetweenallERR1608316-ERR1608325eCommunity-acquired1Paris(3,635,495)[10]3,436,622e3,438,632Range0e1,mean0.2,median0418,832(lpp0256)ERR2216351eERR2216360Travel-associated(Greece)37EUL165(3,474,638)[6]3,332,884e3,334,1630SNPsbetweenallERR2216391eERR2216400Travel-associated20Denovoassembly(3,560,463)3,544,455e3,545,2440SNPsbetweenallERR2216371eERR2216380sepsisCommunity-acquired477Denovoassembly(3,307,866)3,291,099e3,292,328Range0e1,mean0.2,median02,402,083(intergenic)ERR2216381 eERR2216390Travel-associated2287Denovoassembly(3,344,279)3,328,536e3,329,7830SNPsbetweenallERR2216401eERR2216410Community-acquired1522Denovoassembly(3,621,967)3,589,885e3,591,537)0SNPsbetweenallERR2216361eERR2216370.S. David et al. / Clinical Microbiology and Infection 24 (2018) 1020.e1e1020.e4 1020.e2Legionnaires' disease in England (Table 1). Isolates were stored ate80C. DNA was extracted after 48e72 hours of incubation onbuffered charcoal yeast-extract agar at 37C using the Wizard kit(Promega UK, Southampton, UK), eluted in 1 x Tris-EDTA buffer (pH8.0), and quantiﬁed using GloMax (Promega, UK). SBT was under-taken as described previously [3,4]. WGS was performed on Illu-mina X10 with 150-bp paired-end reads. Raw data were submittedto the European Nucleotide Archive (study accession numberPRJEB12239/ERP013693). Individual accession numbers are pro-vided in Table 1.De novo assemblies were generated [6], and MLSTcheck wasused to conﬁrm the sequence type (ST) from them [7]din partic-ular ensuring that at least one of the mompS alleles matched thatcalled by traditional SBT (since this gene is duplicated). Assemblieswere annotated using Prokka v1.11 [8].Single-nucleotide polymorphisms (SNPs) were called for eachisolate by mapping to a reference genome of the same ST using theBurrowseWheeler aligner [9]. Available reference genomesincluded ST1 (Paris), ST23 (EUL 28), ST37 (EUL 165) and ST42 (EUL120) [6,10]. For the rest (ST20, ST477, ST1522 and ST2287), de novoassemblies were used. A pipeline comprising SAMtools, mpileupand BCFtools was used to call SNPs [11]. Various ﬁlters ensuredhigh-accuracy base-calling, and bases were uncalled if any criteriawere not fulﬁlled (reads matching base 4, reads matching baseper strand2, ratio of ﬁrst to second base call0.75, variant quality50, mapping quality 30, strand bias 0.001, map bias 0.001,tail bias 0.001). SNPs identiﬁed in positions where the base wasuncalled (i.e. ‘n’) in more than one isolate per patient werediscarded.To estimate the number of isolates that need to be analysed toobserve all genotypes identiﬁed from a patient, random samplingof between one and ten isolates from each set of ten same-patientisolates was performed 100 times for each number of isolateswithout replacement.Roary [12] was used to determine gene content variation be-tween isolates from the same patient. Pairs of assemblies were alsoaligned and compared using the ‘dnadiff’ tool (v1.3), which is partof the MUMmer package [13].Public Health England holds approvals to process patient-identiﬁable information for the purposes of infectious diseasesurveillance, in accordance with Section 60 of the Health and SocialCare Act 2001. Ethical approval was not required for this study. Thepatient specimens were submitted for Legionella testing, includingculture from microbiology laboratories in England. The Legionelladata used is collated routinely by the Respiratory and SystemicBacteria Section, Public Health England (PHE) as part of the nationalsurveillance in England and Wales.Table1Numberanddetailsofsingle-nucleotidepolymorphisms(SNPatientAgeSexIsolationdate(andnumberofdayspostinfectiononsetthatsamplewasobtained)Clinicalinformati179MJune2015(5)AdmittedtoITU255MMay2015(8)Notprovided355MJune2016(5)Severecommunitacquiredpneumonia,diarrfebrile471MApril2016(6)AdmittedtoITU564MJune2016(3)Respiratoryfailur666FSeptember2016(8)AdmittedtoITU769MJuly2016(5)AdmittedtoITU,pneumonia,sepsis869MAugust2016(2)AdmittedtoITU,975MSeptember2016(7)Notprovided1046MJuly2016(15)NotprovidedST,sequencetype;bp,basepair;ITU,intensivetherapyunitResultsTo investigate the within-host diversity of L. pneumophila, weﬁrst assessed the diversity of STs (as determined by SBT) amongsteach set of ten isolates recovered from ten individual patients withsporadic Legionnaires' disease. In each patient, all ten isolates hadthe same ST (Table 1).The number of SNPs amongst same-patient isolates was thendetermined. The use of a closely related reference genome ensuredthat maximum resolution was achieved and that almost all SNP-based diversity amongst same-patient isolates would be captured.In seven out of ten patients, no SNPs were detected. In two patients,a maximum of one SNP was observed, and in one patient there wasa maximum of two SNPs (Table 1). In each of these three sets inwhich diversity was observed, nine out of ten isolates were iden-tical, and only one isolate differed by one or two SNPs.S. David et al. / Clinical Microbiology and Infection 24 (2018) 1020.e1e1020.e41020.e3Assuming that the full within-host SNP diversity in each set often same-patient isolates was captured, we performed randomsampling of between one and ten isolates, and calculated thenumber of times that the full diversity was captured with thatnumber of isolates. Since no SNPs were found amongst isolatesfrom seven out of ten patients, the mean probability of capturingthe full diversity with only one sample is 70%. This probability risesas the number of samples analysed increases and, on average, sevenisolates are required to have a 90% chance of capturing all geno-types (Fig. 1).Finally, we investigated the extent of gene content variationbetween isolates from the same patient. We found no evidence ofvariation in gene content except for small differences introduced byassembly artefacts.DiscussionThis study provides the most comprehensive analysis of within-host diversity of L. pneumophila in Legionnaires' disease patients todate. The results demonstrate either no or very low within-hostdiversity in ten patients. We also show that, on average, analysisof one isolate provides a 70% chance of capturing all within-hostvariation found with ten isolates. Very low within-host diversityhas also been observed previously [6,14], albeit with lowernumbers of isolates and patients. Others have reported the oppo-site, including Coscolla et al. [15] who reported mixed infections inseveral patients based on SBT proﬁles from uncultured respiratorysamples. Another study that usedWGS identiﬁed two same-patientFig. 1. Boxplots showing the probability that all distinct genotypes found amongst each sanalysed. Probabilities were determined using 100 random samples without replacementbilities for each of the ten patients, and the red squares show the mean across all ten patieisolates belonging to distinct ST191 subtypes that differed byapproximately 20 SNPs [16]. However, multiple isolates from threeother patients in the same study were identical. Thus, while ourstudy suggests that very low within-host diversity is the norm (atleast in sporadic infections), greater diversity has occasionally beenobserved. Indeed, within-host diversity likely depends on severalenvironmental, clinical and epidemiological factors, including thediversity of L. pneumophila in environmental sources, variation ininfectious dose between patients, and the duration of infectionprior to sampling.A signiﬁcant limitation to our study is that the use of culturingprocedures may favour growth of some strains over others, therebyreducing the observed diversity. Furthermore, because of thecollection of isolates from a single time point, as well as the relianceon culture, it is not possible to determine whether the observeddiversity was present at the start of the infection, or whether itevolved during the infection or subsequently in culture. We pro-pose that these limitations may be overcome in future studies bythe use of metagenomics on multiple samples obtained over timefrom the same patient.Transparency declarationJP is a paid consultant to Speciﬁc Technologies LLC. VJC is afﬁl-iated with, and the research was partly supported by, the NationalInstitute for Health Research Health Protection Research Unit (NIHRHPRU) in Respiratory Infections at Imperial College London inpartnership with Public Health England (PHE). This study waset of ten same-patient isolates are observed when between one and ten isolates arefor each number of isolates analysed. The blue circles represent the individual proba-nts.S. David et al. / Clinical Microbiology and Infection 24 (2018) 1020.e1e1020.e4 1020.e4funded by Public Health England and the Wellcome Trust (grantnumber 098051).AcknowledgementsWe would like to thank colleagues for submitting specimens toRVPBRU, and Lalita Vaghji and Jessica Townley for performing theprimary isolation of L. pneumophila.References[1] Rowbotham TJ. Preliminary report on the pathogenicity of Legionella pneu-mophila for freshwater and soil amoebae. J Clin Pathol 1980;33:1179e83.[2] Muder RR, Yu VL, Woo AH. Mode of transmission of Legionella pneumophiladacritical review. Arch Intern Med 1986;146:1607e12.[3] Gaia V, Fry NK, Afshar B, Luck PC, Meugnier H, Etienne J, et al. Consensussequence-based scheme for epidemiological typing of clinical and environ-mental isolates of Legionella pneumophila. J Clin Microbiol 2005;43:2047e52.[4] Ratzow S, Gaia V, Helbig JH, Fry NK, Luck PC. Addition of neuA, the geneencoding N-acylneuraminate cytidylyl transferase, increases the discrimina-tory ability of the consensus sequence-based scheme for typing Legionellapneumophila serogroup 1 strains. J Clin Microbiol 2007;45:1965e8.[5] Mentasti M, Fry NK, Afshar B, Palepou-Foxley C, Naik F, Harrison TG. Appli-cation of Legionella pneumophila-speciﬁc quantitative real-time PCR combinedwith direct ampliﬁcation and sequence-based typing in the diagnosis andepidemiological investigation of Legionnaires' disease. Eur J Clin MicrobiolInfect Dis 2012;31:2017e28.[6] David S, Mentasti M, Tewolde R, Aslett M, Harris SR, Afshar B, et al. Evaluationof an optimal epidemiological typing scheme for Legionella pneumophila withwhole-genome sequence data using validation guidelines. J Clin Microbiol2016;54:2135e48.[7] Page AJ, Taylor B, Keane JA. Multilocus sequence typing by blast from de novoassemblies against PubMLST. JOSS 2016;1:118.[8] Seemann T. Prokka. Rapid prokaryotic genome annotation. Bioinformatics2014;30:2068e9.[9] Li H, Durbin R. Fast and accurate short read alignment with BurrowseWheelertransform. Bioinformatics 2009;25:1754e60.[10] Cazalet C, Rusniok C, Brüggemann H, Zidane N, Magnier A, Ma L, et al. Evi-dence in the Legionella pneumophila genome for exploitation of host cellfunctions and high genome plasticity. Nat Genet 2004;36:1165e73.[11] Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequencealignment/map format and SAMtools. Bioinformatics 2009;25:2078e9.[12] Page A, Cummins C, Hunt M, Wong V, Reuter S, Holden M, et al. Roary: rapidlarge-scale prokaryote pan genome analysis. Bioinformatics 2015;31:3691e3.[13] Kurtz S, Phillippy A, Delcher A, Smoot M, Shumway M, Antonescu C, et al.Versatile and open software for comparing large genomes. Genome Biol2004;5:R12.[14] Bartley PB, Ben Zakour NL, Stanton-Cook M, Muguli R, Prado L, Garnys V, et al.Hospital-wide eradication of a nosocomial Legionella pneumophila serogroup 1outbreak. Clin Infect Dis 2016;62:273e9.[15] Coscolla M, Fernandez C, Colomina J, Sanchez-Buso L, Gonzalez-Candelas F.Mixed infection by Legionella pneumophila in outbreak patients. Int J MedMicrobiol 2014;304:307e13.[16] McAdam PR, Vander Broek CW, Lindsay DS, Ward MJ, Hanson MF, Gillies M,et al. Gene ﬂow in environmental Legionella pneumophila leads to genetic andpathogenic heterogeneity within a Legionnaires' disease outbreak. GenomeBiol 2014;15:504.

Low genomic diversity of Legionella pneumophila within clinical specimens.

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Low genomic diversity of Legionella pneumophila within clinical specimens.

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