Genome Sequencing Links Persistent Outbreak ofLegionellosis in Sydney (New South Wales, Australia) to anEmerging Clone of Legionella pneumophila Sequence Type 211

Verlaine J. Timms,a Rebecca Rockett,a Nathan L. Bachmann,b Elena Martinez,a Qinning Wang,c Sharon C.-A. Chen,c,d

Neisha Jeoffreys,c Peter J. Howard,c Anna Smith,e Sheena Adamson,f Robin Gilmour,f Vicky Sheppeard,f Vitali Sintchenkoa,c,d

aCentre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, New SouthWales, Australia

bCentenary Institute, University of Sydney, Sydney, New South Wales, AustraliacCentre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology andMedical Research, NSW Health Pathology, Sydney, New South Wales, Australia

dMarie Bashir Institute for Infectious Diseases and Biosecurity, Sydney Medical School, The University ofSydney, New South Wales, Australia

eForensic and Analytical Science Services, NSW Health Pathology, Sydney, New South Wales, AustraliafHealth Protection, NSW Health, North Sydney, New South Wales, Australia

ABSTRACT The city of Sydney, Australia, experienced a persistent outbreak of Legion-ella pneumophila serogroup 1 (Lp1) pneumonia in 2016. To elucidate the source andguide public health actions, the genomes of clinical and environmental Lp1 isolates re-covered over 7 weeks were examined. A total of 48 isolates from human cases and cool-ing towers were sequenced and compared using single-nucleotide polymorphism (SNP)-based core-genome multilocus sequencing typing (MLST) and pangenome approaches.All three methods confirmed phylogenetic relatedness between isolates associated withoutbreaks in the Central Business District (CBD) in March and May and those in suburb1. These isolates were designated the “main cluster” and consisted of isolates fromtwo patients from the CBD March outbreak, one patient and one tower isolate fromsuburb 1, and isolates from two cooling towers and three patients from the CBDMay outbreak. All main cluster isolates were sequence type 211 (ST211), which pre-viously has only been reported in Canada. Significantly, pangenome analysis identi-fied mobile genetic elements containing a unique type IV A F-type secretion system(T4ASS), which was specific to the main cluster, and cocirculating clinical strains,suggesting a potential mechanism for increased fitness and persistence of the out-break clone. Genome sequencing enabled linking of the geographically dispersedenvironmental sources of infection among the spatially and temporally coincidingcases of legionellosis in a highly populated urban setting. The discovery of a uniqueT4ASS emphasizes the role of genome recombination in the emergence of success-ful Lp1 clones.

IMPORTANCE A new emerging clone has been responsible for a prolonged legion-ellosis outbreak in Sydney, Australia. The use of whole-genome sequencing linkedtwo outbreaks thought to be unrelated and confirmed the outliers. These findingsled to the resampling and subsequent identification of the source, guiding publichealth actions and bringing the outbreak to a close. Significantly, the outbreak clonewas identified as sequence type 211 (ST211). Our study reports this ST in the South-ern Hemisphere and presents a description of ST211 genomes from both clinical andenvironmental isolates. A unique mobile genetic element containing a type IV secre-tion system was identified in Lp1 ST211 isolates linked to the main cluster and Lp1ST42 isolates that were cocirculating at the time of the outbreak.

Received 14 September 2017 Accepted 27November 2017

Accepted manuscript posted online 15December 2017

Citation Timms VJ, Rockett R, Bachmann NL,Martinez E, Wang Q, Chen SC-A, Jeoffreys N,Howard PJ, Smith A, Adamson S, Gilmour R,Sheppeard V, Sintchenko V. 2018. Genomesequencing links persistent outbreak oflegionellosis in Sydney (New South Wales,Australia) to an emerging clone of Legionellapneumophila sequence type 211. Appl EnvironMicrobiol 84:e02020-17. https://doi.org/10.1128/AEM.02020-17.

Editor Donald W. Schaffner, Rutgers, The StateUniversity of New Jersey

© Crown copyright 2018. The government ofAustralia, Canada, or the UK (“the Crown”) ownsthe copyright interests of authors who aregovernment employees. The Crown Copyrightis not transferable.

Address correspondence to Verlaine J. Timms,Verlaine.timms@health.nsw.gov.au.

PUBLIC AND ENVIRONMENTALHEALTH MICROBIOLOGY

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KEYWORDS Legionella pneumophila, Legionnaires’ disease, prevention and control,molecular epidemiology, bacterial genomics, whole-genome sequencing

Between February and May 2016, Sydney, Australia, experienced several out-breaks of human legionellosis. These outbreaks spanned three precincts within

the Greater Sydney region, which were 18 km apart, lasted over 7 weeks, andincluded the Central Business District (CBD), an area visited by over 610,000 peopleper day. The agent responsible was found to be Legionella pneumophila serogroup1 (Lp1), a ubiquitous microorganism with a natural habitat of water reservoirs whereit is an intracellular parasite of protozoa.

The genus Legionella contains over 60 species. All species are found in the environ-ment, and about half of these can cause a life-threatening illness in humans. L.pneumophila is the major pathogen of this group and is further subdivided into 16serogroups, with the majority (80 to 84%) of legionellosis cases caused by Lp1 (1–3).Since the first description of Legionnaires’ disease in 1976, when a large outbreak ofpneumonia occurred at an American Legion conference in Philadelphia, PA (4), thisinfection has been implicated in multiple sporadic cases and community outbreaksglobally, most commonly in males over 50, people with comorbidities, and smokers (5).

Recombination drives diversity in L. pneumophila (6, 7), providing the species witha suite of mobile genetic elements, including plasmids and pathogenicity islands(8–11). These mobile genetic elements encode virulence and fitness factors, such astype IV secretion systems (T4SSs). The type B T4SSs carry the Dot/Icm genes imperativefor intracellular replication and survival in both amoebae and human macrophages (12,13), while the type A T4SSs possess a Tra region and can be found on both plasmidsand chromosomes. Gene sequencing-based typing (SBT) methods have been used forpublic health laboratory surveillance (14); however, the limited discrimination power ofSBT had led to a wider application of high-resolution whole-genome sequencing (WGS)in the investigation of community outbreaks of legionellosis. WGS technological ad-vancements now provide increased discrimination of outbreak isolates, especially forendemic clones, such as ST1 (15–17). This study aimed to elucidate the source andincreased persistence of the prolonged Sydney 2016 outbreak by examining thegenomes of clinical and environmental Lp1 isolates collected over that period.

RESULTS

Four outbreaks of severe community-acquired pneumonia in adults with positiveurinary antigen for Lp1 were identified in metropolitan Sydney in the first half of 2016;three of these outbreaks were found to be linked. Nine human cases were epidemio-logically associated with the CBD March outbreak and six cases in the CBD Mayoutbreak, all with common exposure links to the Sydney CBD. Detailed epidemiologicalinformation on the CBD cases can be found in a public health investigation report (18).Four cases were linked in April to suburb 1. Also, in May, five cases were linked tosuburb 2. One case (case 12) in May was linked to both suburb 2 and the CBD clusters(Table 1). Lp1 was cultured from respiratory samples from 12 patients and from 14cooling towers. All clinical and environmental isolates collected, together with twocontrol strains and two Lp1 isolates obtained from epidemiologically unrelated cases,were subjected to genome sequencing. All 48 genomes were de novo assembled, andtheir genome sizes ranged between 3.2 and 3.4 Mb, with a G�C content of 39%. Thecore genome, as determined by the Roary pipeline, consisted of 2,181 genes. Theaccessory genome had 2,273 genes out of a total of 5,919 genes (including 99 soft-coregenes and 1,366 shell genes that were not counted in the core and accessory,respectively). The numbers of accessory genes were compared between the two majorclades of sequence type 1 (ST1) and ST211. The ST1 strains contained an accessorygenome of 42 genes, and the ST211 strains were found to have 79 genes in theaccessory genome, despite there being more total genes in the ST1 group (n � 3,421)than in the ST211 group (n � 3,179).

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Genome comparisons based on single-nucleotide polymorphism (SNP)-based map-ping, core-genome multilocus sequence typing (cgMLST), and pangenome analysisproduced congruent results and confirmed phylogenetic relatedness between isolatesassociated with outbreaks in the CBD (March and May) and suburb 1 (Fig. 1 and 2; see

FIG 1 SNP-based mapping phylogeny of all outbreak isolates between February and May 2016 in Sydney. A maximum likelihood phylogenetic tree was builtusing 204,241 SNPs relative to L. pneumophila strain Philadelphia. The top scale indicates branch length in number of SNPs. The main cluster is outlined in anorange box and expanded; therefore, branches are not to scale in the inset. Clinical and environmental isolates are distinguished by “C” or “E,” as per the legend.Isolates from each of the different outbreaks are color-coded according to the legend. The main cluster contains isolates from cases of CBD March; case 1 (BC1,BC2, and BC7) and case 3 (BC5); suburb 1, case 5 (RC1 and RC2) and tower 11 (isolates RE4 to RE7); CBD May, cases 6 (BC6), 7 (BC3 and BC4), and 8 (BC8), andtowers 14 (BE1) and 15 (BE2).

FIG 2 The pangenome of the complete Sydney 2016 Lp1 data set (n � 48). (A) Maximum likelihood tree showing the main cluster within clade 3. (B) Pangenomesorted from core genes on the left to accessory genes to the right. (C) Heatmap showing presence (blue) and absence (white) of genomic regions. The uniquegenomic island (including the T4ASS) present in the main cluster (region II), the outlier (BC9 and BC10 in blue), and related environmental strains (BE3 and BE4in blue) are indicated by red circles. Region I (green circles) demonstrates genes that were unique to clade 3, including reference isolates and the main cluster.Region III (orange circles) indicates genes that were unique to the main cluster and related environmental strains BE3 and BE4 only. The image was preparedusing Phandango (40).

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also Fig. S1 in the supplemental material), and these isolates were designated the “maincluster.” The CBD March outbreak showed that isolates from two patients, case 1(isolates BC1, BC2, and BC7) and case 3 (isolate BC5), clustered together (Table 1 andFig. 1 and 2). For the next outbreak in suburb 1, case 5 isolates (RC1 and RC2) clusteredwith tower 11 isolates (RE4 to RE7), and in the CBD May outbreak, isolates from tower14 (BE1) and tower 15 (BE2) and case 6 (BC6), case 7 (BC3 and BC4), and case 8 (BC8)were from the same genetic cluster. All isolates from the main cluster were also typedas ST211 (Table 1). Isolates from cases 9 to 12 (GC1 to GC4) were all linked to suburb2 and, along with case 2 (isolates BC9 and BC10) from CBD March and case 4 (isolateRC3) from suburb 1 outbreaks, appeared to be genomically distinct and not related tothe main cluster (Table 1 and Fig. 1 and 2). The maximum SNP difference between maincluster isolates was 87 SNPs between CBD tower isolates and suburb 1 clinical isolates.There were 44 SNP differences between suburb 1 tower isolates and suburb 1 clinicalisolates. Among all clinical isolates from the main cluster, there was an averagedifference of 66 SNPs, and repeated isolates from the same case of human diseasediffered by fewer than 45 SNPs. The two towers harboring environmental Lp1 ST211strains were approximately 800 m apart on different buildings.

Pangenome analysis further revealed three regions of interest related to the maincluster (Fig. 2). The first (region I) was a large region of 178 genes that was present inclade 3 and included the main cluster. Region I was also found in all reference strains,the two past outbreak isolates, closely related environmental strains BE3 and BE4, andan outlier, isolate GC4. Region I included the genes for Dot/Icm, luxR, Ankyrin repeats,and beta-lactamase, among others, indicating that this was a type IV B secretion system(T4BSS). The second region (region II) consisted of 58 genes that were unique to themain cluster, case 2 isolates BC9 and BC10, which were circulating at the same time, andtwo closely related environmental isolates, BE3 and BE4 (Fig. 2). Region II was found tocarry another T4SS, an A F-type secretion system (T4ASS), and this region had a higherG�C content of 42% than the rest of the genome (Table S1). This region, designatedT4ASS-Sydney, had between 99 and 100% homology to similar conjugative elements inonly four other L. pneumophila Lp1 strains, two from closed genomes, those of LPE509(GenBank accession no. NC_020521.1) (Fig. 3 and S2) and C1-S (GenBank accession no.CP015932.1), and two further clinical isolates, with one ST37 isolate obtained in 2003 inthe United Kingdom (GenBank accession no. LT632617.1), and the other an ST42 clinicalisolate from Germany (GenBank accession no. LT632616.1). T4ASS-Sydney is 73 kb long,with an insertion site adjacent to three tRNA genes (tRNAArg, tRNALys, and tRNALys) (Fig.S2). Further analysis revealed that T4ASS-Sydney was present in one copy and insertedadjacent to the T4ASS homologous to that in strain Philadelphia. The T4SSs of theST211 genomes were more similar to the Philadelphia strain than were the T4SSs of L.pneumophila strains Paris and 130b, and this observation was also backed by thephylogeny shown in Fig. 1. The genomes of environmental strains BE1 and LPE509 andthe clinical isolate BC9 (ST42) did not contain the Philadelphia-like T4ASS; only T4ASS-Sydney was found in these strains (Fig. 3). Another smaller region was unique to themain cluster and closely related isolates BE3 and BE4, as well as Lp1 strain Philadelphia.This region, denoted region III, contained mainly hypothetical proteins, acyltransferasegenes, and a putative multidrug export ATP-binding/permease protein (Table S1).

DISCUSSION

Our findings indicate that cases among four temporally and/or spatially separateoutbreaks of legionellosis that occurred in quick succession in metropolitan Sydney in2016 were caused by a common clone of Lp1 ST211. The geographical distancebetween sites of potential exposure was much larger than previously recognized (Knoxet al. [19]), and this coupled with the temporal differences between these outbreaks ledto the initial assumption that they were related to distinct exposures resulting fromseparate breaches of environmental health controls. However, phylogenetic analysis onboth clinical and environmental isolates from these outbreaks by three independentgenomic approaches confirmed that a single Lp1 clone was common to three of the

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four outbreaks. The CBD March outbreak was consonant with the suburb 1 outbreak 18km away, and both of these were genetically related to the CBD May outbreak thatoccurred 6 weeks later. Factors, such as cooling tower maintenance and other envi-ronmental considerations, may have contributed to this wave of Lp1 disease. Ourfindings indicate that SNP-based and core-genome comparisons of genome related-ness and a pangenome-based analysis in the investigation of Lp1 outbreaks (20) havecomparable discrimination power and should be supplemented by an analysis ofmobile genetic elements given the high recombination rates in Lp1 (6, 21).

This study reports the presence of fully sequenced Lp1 ST211 in the Southern

FIG 3 Nucleotide comparison of T4SSs from selected clinical and environmental strains from the Sydney 2016 outbreak compared to those in L. pneumophilastrains Paris, 130b, Philadelphia 1, and LPE509. Gray vertical shading blocks indicate regions of shared similarity, according to BLASTn. Colored pointersrepresent T4SS genes, where the functions have been inferred from BLAST searches and annotation by Prokka. The type of T4SS is indicated by color accordingto the legend. The image was prepared using Easyfig (41).

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Hemisphere. This ST has not been reported in the United States or Europe and wasthought to reside exclusively in the colder climate of Canada, where it was firstidentified in 1989 using sequence-based typing (22). In Ontario, Canada, ST211 wasfound to be responsible for 12.5% of human cases and has since become one of themost persistent and predominant STs, almost completely replacing the previouslydominant Lp1 ST1. There is also a suggestion that ST1 may be the predominant ST inAustralia (17), and further surveillance of ST211 and ST1 clones in Sydney is warranted.The last Lp1 outbreak in Sydney was over 10 years ago and was dominated by an ST1clone (23). In addition to the assessment of potential relationships between clinicalcases and environmental sources, WGS of Legionella isolates enables the examinationof diversity and genomic structures that may have augmented their persistence in theenvironment (24). Our pangenome analysis revealed a unique conjugative Tra (F-type)element carrying a T4ASS, and this was present in all outbreak isolates; more signifi-cantly, however, it was also found in closely related environmental isolates and ST42isolates that infected case 2 at the same time as the CBD March outbreak. Interestingly,this element had 99% nucleotide homology to a T4ASS found in four previouslyreported clinical strains from the United States and Europe and an environmentalisolate from Japan, none of which are ST211 but are STs in the top five of outbreak-causing clones (25).

The T4SSs of Legionella are found in both the core and accessory genome, and someare known to contribute to fitness and virulence, playing a crucial role in intracellularreplication and survival (9, 13, 26, 27). Unique T4ASS have been found in previousoutbreak isolates (17), including from a recent outbreak in western Canada, which isunique in its dry cold conditions that were initially thought to be too harsh for thesurvival of L. pneumophila (19). The element described in this study contained geneshomologous to the Lvh region of other L. pneumophila strains, and genes from thisregion are thought to assist in intracellular replication (28, 29). T4ASS-Sydney was foundin only one copy, adjacent to another T4ASS with high homology to that described inL. pneumophila strain Philadelphia (30). However, it was also observed in strains withoutthe Philadelphia T4ASS, suggesting that it has been acquired independently. It ispossible that these genomic features reflect high recombination potential and fitnessof Lp1 ST211 and help explain its emergence as a highly successful outbreak clone.Earlier reports attempted to define outbreak strains from nonoutbreak strains, andgenome sequencing has identified recombination as a major contributor to Lp1variability which occurs across all strains, although some STs appear to have differentrecombination rates (6). In conclusion, SNP-based, core-genome-based, and pangenome-based analyses of Lp1 isolates can assist in deciphering and confirming transmissionpathways during the investigation of complex outbreaks of legionellosis. Comparativegenomics of clinical and environmental isolates of Lp1 suggest that the emergingST211 clone was responsible for three sequential outbreaks of legionellosis in metro-politan Sydney separated geographically and temporally over 7 weeks. Mobile geneticelements that included a T4ASS in the Lp1 ST211 and cocirculating strains may haveaugmented the fitness and persistence of the outbreak clone in the environment.

MATERIALS AND METHODSBacterial isolates and serogrouping. Between February and May 2016, the community outbreaks

of legionellosis were identified by positive urinary Lp1 antigen results and/or respiratory tract culturespositive for Lp1. Clinical and environmental Lp1 isolates from three geographically distinct locations, theCentral Business District (CBD), 18 km south of the CBD (suburb 1), and 12 km west of the CBD (suburb2), were studied (Table 1 and Fig. 4). Isolates from human cases epidemiologically linked to the CBD werecultured from samples collected from 7 to 15 March 2016 (CBD March outbreak) and from 28 April to the11 May 2016 (CBD May outbreak). There was a period of 6 weeks where no cases from the CBD weredetected between March and April. Environmental isolates from cooling towers were referred from theForensic and Analytical Science Service, while clinical isolates were cultured from sputum samples orbronchoalveolar lavage fluid referred to the Centre for Infectious Diseases and Microbiology LaboratoryServices, Westmead Hospital, Sydney. Isolates were grown on buffered charcoal-yeast extract (BCYE)agar, incubated at 37°C for up to 7 days, and identified by standard phenotypic characteristics (31).Serogrouping of each isolate was performed using the Legionella latex test (Oxoid, Thermo FisherScientific, North Ryde, NSW, Australia).

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Clinical and environmental isolates alongside the type strains L. pneumophila ATCC 33152 (Philadel-phia; GenBank accession no. AE017354) and L. pneumophila ATCC 33215 (Chicago 2; BioProject accessionno. PRJNA239272) were grown in the laboratory, DNA was extracted, and sequencing libraries wereprepared alongside all the other isolates as controls of assembly- and bioinformatics-based pipelines. Inaddition, two Lp1 clinical isolates from previous unrelated outbreaks in Sydney (Reference 2013 andReference 2015) were included as unrelated outgroups.

DNA extraction and WGS. Genomic DNA was extracted from pure cultures using the DNeasy bloodand tissue kit (Qiagen, Chadstone, Victoria, Australia), with a 3-h proteinase K digestion at 56°C. Thequality of each DNA sample was measured using a NanoDrop ND-1000 spectrophotometer (NanoDropTechnologies, Thermo Fisher Scientific), and the 260/280 nm and 260/230 nm ratios were inspected. DNApurity was determined to be satisfactory if the 260/280 ratio fell between 1.6 and 2.2 and the 260/230ratio fell between 1.8 and 2.2. Paired-end indexed libraries of 150 bp in length were prepared from aninput of 1 ng of purified DNA with the Nextera XT library preparation kit (Illumina, Scoresby, Victoria,Australia), as per the manufacturer’s instruction. DNA libraries were then sequenced using the NextSeq500 (Illumina).

TABLE 1 Legionella pneumophila serogroup 1 isolates included in this study

Isolate identification no. Origin Case no. in outbreak Association with locationa SBTb

BC7 Clinical Case 1 CBD March 211BC10 Clinical Case 2 CBD March 42BC5 Clinical Case 3 CBD March 211Reference control 1 Clinical ATCC 33152Reference control 2 Clinical ATCC 33215BC2 Clinical Case 1 CBD March 211BC1 Clinical Case 1 CBD March 211BC9 Clinical Case 2 CBD March 42Reference 2013 Clinical Reference case 1 Not related 762Reference 2015 Clinical Reference case 2 Not related NDBE3 Environmental Tower 1 CBD March NDBE11 Environmental Tower 2 CBD March 1BE12 Environmental Tower 3 CBD March 1BE13 Environmental Tower 4 CBD March 1BE7 Environmental Tower 5 CBD March 1BE5 Environmental Tower 6 CBD March 1BE17 Environmental Tower 6 CBD March 1BE20 Environmental Tower 7 CBD March 284BE19 Environmental Tower 8 CBD March 1BE14 Environmental Tower 9 CBD March 1BE10 Environmental Tower 9 CBD March 1BE6 Environmental Tower 9 CBD March 1BE8 Environmental Tower 10 CBD March 1RC3 Clinical Case 4 Suburb 1 NDRC1 Clinical Case 5 Suburb 1 211RC2 Clinical Case 5 Suburb 1 211RE6 Environmental Tower 11 Suburb 1 211RE4 Environmental Tower 11 Suburb 1 211RE7 Environmental Tower 11 Suburb 1 211RE5 Environmental Tower 11 Suburb 1 211RE18 Environmental Tower 12 Suburb 1 1RE2 Environmental Tower 12 Suburb 1 1RE16 Environmental Tower 12 Suburb 1 1RE15 Environmental Tower 12 Suburb 1 1RE3 Environmental Tower 12 Suburb 1 1RE1 Environmental Tower 10 Suburb 1 284BC6 Clinical Case 6 CBD May 211BC4 Clinical Case 7 CBD May 211BC3 Clinical Case 7 CBD May 211BC8 Clinical Case 8 CBD May 211BE4 Environmental Tower 13 CBD May NDBE1 Environmental Tower 14 CBD May 211BE9 Environmental Tower 9 CBD May 1BE2 Environmental Tower 15 CBD May 211GC3 Clinical Case 9 Suburb 2 1983GC2 Clinical Case 10 Suburb 2 NDGC1 Clinical Case 11 Suburb 2 84GC4 Clinical Case 12 Suburb 2/CBD May NDaCBD March, isolates from the Central Business District during February to March 2016; CBD May, isolates from the Central Business District between April and May2016.

bSBT, sequence-based type; ND, not determined due to one or more alleles being new or a combination of alleles being new.

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Genome assembly and analysis. FASTQ files were imported into Geneious (version 8.0.4) andmapped to a curated reference of L. pneumophila Philadelphia (GenBank accession no. NC_002942) usingthe bwa plugin (version 0.7.10) with bacteriophage, insertion sequences, and other repeat regionsremoved, as described by Coil et al. (32). Two more reference genomes were added to the phylogeny,those of L. pneumophila strain Paris (GenBank accession no. NC_006368.1) and L. pneumophila strainLorraine (BioProject accession no. PRJNA67921). Quality-based variant detection was performed usingCLC Genomics Workbench version 7.0 (CLC bio, Aarhus, Denmark). Variant detection thresholds were setfor a minimum coverage of 10-fold and minimum variant frequency of 75%. SNPs were excluded if theywere in regions with a minimum fold coverage of �10-fold, within 10 bp of another SNP, or within �15bp from the end of a contig. Maximum likelihood phylogenetic trees were constructed from SNP matricesusing the general time-reversible (GTR) model with 100 bootstrap replications.

Sequencing reads were assembled with SPAdes (33) and annotated with Prokka (34). Sequence-based typing (SBT) was performed with seven loci by uploading identified alleles to the database andobtaining locus numbers (14). In addition, core-genome multilocus sequence typing (cgMLST) wasconducted using the Ridom SeqSphere software (Qiagen) employing gene definitions determined fromclosed or complete L. pneumophila genomes from NCBI GenBank (35). Using these criteria, the coregenome was determined to be 1,530 genes, with an accessory genome of 1,370 genes. Furtherpangenome assessment and visualization were performed using the Roary pipeline (36) that includesalignment using MAFFT (37) and tree building with FastTree (38). Specific protein and nucleotidecomparisons were made (including the unique T4ASS) to all available Legionella genomes on NCBI usingthe relevant BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Additional analysis on the unique T4SS described in this study required a comparison with tworecently sequenced (and closed) genomes, those of LPE509 (GenBank accession no. NC_020521.1) andC1-S (GenBank accession no. CP015932.1). L. pneumophila strain Philadelphia was also included in thesecomparisons, given that it also contains a T4ASS that is of similar size and has the same insertion site asthe T4ASS described in this study. Pairwise genome comparisons of the unique T4ASS were performedusing BLASTn and visualized using Artemis (39), Easyfig (41), and Phandango (40). The presence of allcharacterized T4SSs of L. pneumophila was determined and compared: LGI-1, LGI-2, Lvh, P-type, F-type,and Dot/Icm in eight strains, including two environmental strains (one of which was ST211) and threeclinical strains (two of which were ST211). The genomes of L. pneumophila strain Paris and L. pneumophilastrain 130b (GenBank accession no. NZ_CAFM01000092) were also included to enable a comparison withall T4SSs in L. pneumophila.

Accession number(s). The genomic data have been deposited in the NCBI Sequence Read Archive(SRA) (http://www.ncbi.nlm.nih.gov/Traces/sra/) under study accession number SRP117289.

FIG 4 Map of metropolitan Sydney indicating locations of the outbreak during 2016. The red circlesindicate suburb 2, CBD, and suburb 1 and mark the approximate case clustering in these areas. (Adaptedwith permission from the NSW Department of Industry.)

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02020-17.

SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.

ACKNOWLEDGMENTSWe thank public health professionals and epidemiologists from NSW Health and the

City of Sydney, as well as laboratory scientists from NSW Health Pathology for providingclinical and environmental isolates and their assistance in collecting epidemiologicaland environmental information.

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