ABSTRACT
The city of Sydney, Australia, experienced a persistent outbreak of Legionella pneumophila serogroup 1 (Lp1) pneumonia in 2016. To elucidate the source and guide public health actions, the genomes of clinical and environmental Lp1 isolates recovered over 7 weeks were examined. A total of 48 isolates from human cases and cooling 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 with outbreaks in the Central Business District (CBD) in March and May and those in suburb 1. These isolates were designated the “main cluster” and consisted of isolates from two patients from the CBD March outbreak, one patient and one tower isolate from suburb 1, and isolates from two cooling towers and three patients from the CBD May outbreak. All main cluster isolates were sequence type 211 (ST211), which previously has only been reported in Canada. Significantly, pangenome analysis identified 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 outbreak clone. Genome sequencing enabled linking of the geographically dispersed environmental sources of infection among the spatially and temporally coinciding cases of legionellosis in a highly populated urban setting. The discovery of a unique T4ASS emphasizes the role of genome recombination in the emergence of successful Lp1 clones.
IMPORTANCE A new emerging clone has been responsible for a prolonged legionellosis outbreak in Sydney, Australia. The use of whole-genome sequencing linked two outbreaks thought to be unrelated and confirmed the outliers. These findings led to the resampling and subsequent identification of the source, guiding public health actions and bringing the outbreak to a close. Significantly, the outbreak clone was identified as sequence type 211 (ST211). Our study reports this ST in the Southern Hemisphere and presents a description of ST211 genomes from both clinical and environmental isolates. A unique mobile genetic element containing a type IV secretion system was identified in Lp1 ST211 isolates linked to the main cluster and Lp1 ST42 isolates that were cocirculating at the time of the outbreak.
KEYWORDS: Legionella pneumophila, Legionnaires' disease, prevention and control, molecular epidemiology, bacterial genomics, whole-genome sequencing
INTRODUCTION
Between February and May 2016, Sydney, Australia, experienced several outbreaks of human legionellosis. These outbreaks spanned three precincts within the Greater Sydney region, which were 18 km apart, lasted over 7 weeks, and included the Central Business District (CBD), an area visited by over 610,000 people per day. The agent responsible was found to be Legionella pneumophila serogroup 1 (Lp1), a ubiquitous microorganism with a natural habitat of water reservoirs where it is an intracellular parasite of protozoa.
The genus Legionella contains over 60 species. All species are found in the environment, 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 16 serogroups, 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 of pneumonia occurred at an American Legion conference in Philadelphia, PA (4), this infection has been implicated in multiple sporadic cases and community outbreaks globally, most commonly in males over 50, people with comorbidities, and smokers (5).
Recombination drives diversity in L. pneumophila (6, 7), providing the species with a suite of mobile genetic elements, including plasmids and pathogenicity islands (8–11). These mobile genetic elements encode virulence and fitness factors, such as type IV secretion systems (T4SSs). The type B T4SSs carry the Dot/Icm genes imperative for 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 plasmids and chromosomes. Gene sequencing-based typing (SBT) methods have been used for public health laboratory surveillance (14); however, the limited discrimination power of SBT had led to a wider application of high-resolution whole-genome sequencing (WGS) in the investigation of community outbreaks of legionellosis. WGS technological advancements now provide increased discrimination of outbreak isolates, especially for endemic clones, such as ST1 (15–17). This study aimed to elucidate the source and increased persistence of the prolonged Sydney 2016 outbreak by examining the genomes of clinical and environmental Lp1 isolates collected over that period.
RESULTS
Four outbreaks of severe community-acquired pneumonia in adults with positive urinary 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 epidemiologically associated with the CBD March outbreak and six cases in the CBD May outbreak, all with common exposure links to the Sydney CBD. Detailed epidemiological information 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 to suburb 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 14 cooling towers. All clinical and environmental isolates collected, together with two control strains and two Lp1 isolates obtained from epidemiologically unrelated cases, were subjected to genome sequencing. All 48 genomes were de novo assembled, and their genome sizes ranged between 3.2 and 3.4 Mb, with a G+C content of 39%. The core genome, as determined by the Roary pipeline, consisted of 2,181 genes. The accessory genome had 2,273 genes out of a total of 5,919 genes (including 99 soft-core genes 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 major clades of sequence type 1 (ST1) and ST211. The ST1 strains contained an accessory genome of 42 genes, and the ST211 strains were found to have 79 genes in the accessory genome, despite there being more total genes in the ST1 group (n = 3,421) than in the ST211 group (n = 3,179).
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 | 211 |
| BC10 | Clinical | Case 2 | CBD March | 42 |
| BC5 | Clinical | Case 3 | CBD March | 211 |
| Reference control 1 | Clinical | ATCC 33152 | ||
| Reference control 2 | Clinical | ATCC 33215 | ||
| BC2 | Clinical | Case 1 | CBD March | 211 |
| BC1 | Clinical | Case 1 | CBD March | 211 |
| BC9 | Clinical | Case 2 | CBD March | 42 |
| Reference 2013 | Clinical | Reference case 1 | Not related | 762 |
| Reference 2015 | Clinical | Reference case 2 | Not related | ND |
| BE3 | Environmental | Tower 1 | CBD March | ND |
| BE11 | Environmental | Tower 2 | CBD March | 1 |
| BE12 | Environmental | Tower 3 | CBD March | 1 |
| BE13 | Environmental | Tower 4 | CBD March | 1 |
| BE7 | Environmental | Tower 5 | CBD March | 1 |
| BE5 | Environmental | Tower 6 | CBD March | 1 |
| BE17 | Environmental | Tower 6 | CBD March | 1 |
| BE20 | Environmental | Tower 7 | CBD March | 284 |
| BE19 | Environmental | Tower 8 | CBD March | 1 |
| BE14 | Environmental | Tower 9 | CBD March | 1 |
| BE10 | Environmental | Tower 9 | CBD March | 1 |
| BE6 | Environmental | Tower 9 | CBD March | 1 |
| BE8 | Environmental | Tower 10 | CBD March | 1 |
| RC3 | Clinical | Case 4 | Suburb 1 | ND |
| RC1 | Clinical | Case 5 | Suburb 1 | 211 |
| RC2 | Clinical | Case 5 | Suburb 1 | 211 |
| RE6 | Environmental | Tower 11 | Suburb 1 | 211 |
| RE4 | Environmental | Tower 11 | Suburb 1 | 211 |
| RE7 | Environmental | Tower 11 | Suburb 1 | 211 |
| RE5 | Environmental | Tower 11 | Suburb 1 | 211 |
| RE18 | Environmental | Tower 12 | Suburb 1 | 1 |
| RE2 | Environmental | Tower 12 | Suburb 1 | 1 |
| RE16 | Environmental | Tower 12 | Suburb 1 | 1 |
| RE15 | Environmental | Tower 12 | Suburb 1 | 1 |
| RE3 | Environmental | Tower 12 | Suburb 1 | 1 |
| RE1 | Environmental | Tower 10 | Suburb 1 | 284 |
| BC6 | Clinical | Case 6 | CBD May | 211 |
| BC4 | Clinical | Case 7 | CBD May | 211 |
| BC3 | Clinical | Case 7 | CBD May | 211 |
| BC8 | Clinical | Case 8 | CBD May | 211 |
| BE4 | Environmental | Tower 13 | CBD May | ND |
| BE1 | Environmental | Tower 14 | CBD May | 211 |
| BE9 | Environmental | Tower 9 | CBD May | 1 |
| BE2 | Environmental | Tower 15 | CBD May | 211 |
| GC3 | Clinical | Case 9 | Suburb 2 | 1983 |
| GC2 | Clinical | Case 10 | Suburb 2 | ND |
| GC1 | Clinical | Case 11 | Suburb 2 | 84 |
| GC4 | Clinical | Case 12 | Suburb 2/CBD May | ND |
CBD March, isolates from the Central Business District during February to March 2016; CBD May, isolates from the Central Business District between April and May 2016.
SBT, sequence-based type; ND, not determined due to one or more alleles being new or a combination of alleles being new.
Genome comparisons based on single-nucleotide polymorphism (SNP)-based mapping, core-genome multilocus sequence typing (cgMLST), and pangenome analysis produced congruent results and confirmed phylogenetic relatedness between isolates associated with outbreaks in the CBD (March and May) and suburb 1 (Fig. 1 and 2; see also Fig. S1 in the supplemental material), and these isolates were designated the “main cluster.” 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 and Fig. 1 and 2). For the next outbreak in suburb 1, case 5 isolates (RC1 and RC2) clustered with tower 11 isolates (RE4 to RE7), and in the CBD May outbreak, isolates from tower 14 (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 typed as ST211 (Table 1). Isolates from cases 9 to 12 (GC1 to GC4) were all linked to suburb 2 and, along with case 2 (isolates BC9 and BC10) from CBD March and case 4 (isolate RC3) from suburb 1 outbreaks, appeared to be genomically distinct and not related to the main cluster (Table 1 and Fig. 1 and 2). The maximum SNP difference between main cluster 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 clinical isolates. Among all clinical isolates from the main cluster, there was an average difference of 66 SNPs, and repeated isolates from the same case of human disease differed by fewer than 45 SNPs. The two towers harboring environmental Lp1 ST211 strains were approximately 800 m apart on different buildings.
FIG 1.
SNP-based mapping phylogeny of all outbreak isolates between February and May 2016 in Sydney. A maximum likelihood phylogenetic tree was built using 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 an orange 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), and towers 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) Pangenome sorted from core genes on the left to accessory genes to the right. (C) Heatmap showing presence (blue) and absence (white) of genomic regions. The unique genomic island (including the T4ASS) present in the main cluster (region II), the outlier (BC9 and BC10 in blue), and related environmental strains (BE3 and BE4 in 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 prepared using Phandango (40).
Pangenome analysis further revealed three regions of interest related to the main cluster (Fig. 2). The first (region I) was a large region of 178 genes that was present in clade 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, and an 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 the main cluster, case 2 isolates BC9 and BC10, which were circulating at the same time, and two closely related environmental isolates, BE3 and BE4 (Fig. 2). Region II was found to carry another T4SS, an A F-type secretion system (T4ASS), and this region had a higher G+C content of 42% than the rest of the genome (Table S1). This region, designated T4ASS-Sydney, had between 99 and 100% homology to similar conjugative elements in only 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 in the United Kingdom (GenBank accession no. LT632617.1), and the other an ST42 clinical isolate 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 inserted adjacent to the T4ASS homologous to that in strain Philadelphia. The T4SSs of the ST211 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 the phylogeny shown in Fig. 1. The genomes of environmental strains BE1 and LPE509 and the 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 the main cluster and closely related isolates BE3 and BE4, as well as Lp1 strain Philadelphia. This region, denoted region III, contained mainly hypothetical proteins, acyltransferase genes, and a putative multidrug export ATP-binding/permease protein (Table S1).
FIG 3.
Nucleotide comparison of T4SSs from selected clinical and environmental strains from the Sydney 2016 outbreak compared to those in L. pneumophila strains Paris, 130b, Philadelphia 1, and LPE509. Gray vertical shading blocks indicate regions of shared similarity, according to BLASTn. Colored pointers represent T4SS genes, where the functions have been inferred from BLAST searches and annotation by Prokka. The type of T4SS is indicated by color according to the legend. The image was prepared using Easyfig (41).
DISCUSSION
Our findings indicate that cases among four temporally and/or spatially separate outbreaks of legionellosis that occurred in quick succession in metropolitan Sydney in 2016 were caused by a common clone of Lp1 ST211. The geographical distance between sites of potential exposure was much larger than previously recognized (Knox et al. [19]), and this coupled with the temporal differences between these outbreaks led to the initial assumption that they were related to distinct exposures resulting from separate breaches of environmental health controls. However, phylogenetic analysis on both clinical and environmental isolates from these outbreaks by three independent genomic approaches confirmed that a single Lp1 clone was common to three of the four outbreaks. The CBD March outbreak was consonant with the suburb 1 outbreak 18 km away, and both of these were genetically related to the CBD May outbreak that occurred 6 weeks later. Factors, such as cooling tower maintenance and other environmental considerations, may have contributed to this wave of Lp1 disease. Our findings indicate that SNP-based and core-genome comparisons of genome relatedness and a pangenome-based analysis in the investigation of Lp1 outbreaks (20) have comparable discrimination power and should be supplemented by an analysis of mobile genetic elements given the high recombination rates in Lp1 (6, 21).
This study reports the presence of fully sequenced Lp1 ST211 in the Southern Hemisphere. This ST has not been reported in the United States or Europe and was thought to reside exclusively in the colder climate of Canada, where it was first identified in 1989 using sequence-based typing (22). In Ontario, Canada, ST211 was found to be responsible for 12.5% of human cases and has since become one of the most persistent and predominant STs, almost completely replacing the previously dominant Lp1 ST1. There is also a suggestion that ST1 may be the predominant ST in Australia (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 ST1 clone (23). In addition to the assessment of potential relationships between clinical cases and environmental sources, WGS of Legionella isolates enables the examination of diversity and genomic structures that may have augmented their persistence in the environment (24). Our pangenome analysis revealed a unique conjugative Tra (F-type) element carrying a T4ASS, and this was present in all outbreak isolates; more significantly, however, it was also found in closely related environmental isolates and ST42 isolates 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 previously reported clinical strains from the United States and Europe and an environmental isolate 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 some are known to contribute to fitness and virulence, playing a crucial role in intracellular replication and survival (9, 13, 26, 27). Unique T4ASS have been found in previous outbreak isolates (17), including from a recent outbreak in western Canada, which is unique in its dry cold conditions that were initially thought to be too harsh for the survival of L. pneumophila (19). The element described in this study contained genes homologous to the Lvh region of other L. pneumophila strains, and genes from this region are thought to assist in intracellular replication (28, 29). T4ASS-Sydney was found in only one copy, adjacent to another T4ASS with high homology to that described in L. pneumophila strain Philadelphia (30). However, it was also observed in strains without the Philadelphia T4ASS, suggesting that it has been acquired independently. It is possible that these genomic features reflect high recombination potential and fitness of Lp1 ST211 and help explain its emergence as a highly successful outbreak clone. Earlier reports attempted to define outbreak strains from nonoutbreak strains, and genome sequencing has identified recombination as a major contributor to Lp1 variability which occurs across all strains, although some STs appear to have different recombination rates (6). In conclusion, SNP-based, core-genome-based, and pangenome-based analyses of Lp1 isolates can assist in deciphering and confirming transmission pathways during the investigation of complex outbreaks of legionellosis. Comparative genomics of clinical and environmental isolates of Lp1 suggest that the emerging ST211 clone was responsible for three sequential outbreaks of legionellosis in metropolitan Sydney separated geographically and temporally over 7 weeks. Mobile genetic elements that included a T4ASS in the Lp1 ST211 and cocirculating strains may have augmented the fitness and persistence of the outbreak clone in the environment.
MATERIALS AND METHODS
Bacterial 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 cultures positive for Lp1. Clinical and environmental Lp1 isolates from three geographically distinct locations, the Central Business District (CBD), 18 km south of the CBD (suburb 1), and 12 km west of the CBD (suburb 2), were studied (Table 1 and Fig. 4). Isolates from human cases epidemiologically linked to the CBD were cultured from samples collected from 7 to 15 March 2016 (CBD March outbreak) and from 28 April to the 11 May 2016 (CBD May outbreak). There was a period of 6 weeks where no cases from the CBD were detected between March and April. Environmental isolates from cooling towers were referred from the Forensic and Analytical Science Service, while clinical isolates were cultured from sputum samples or bronchoalveolar lavage fluid referred to the Centre for Infectious Diseases and Microbiology Laboratory Services, 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 Fisher Scientific, North Ryde, NSW, Australia).
FIG 4.
Map of metropolitan Sydney indicating locations of the outbreak during 2016. The red circles indicate suburb 2, CBD, and suburb 1 and mark the approximate case clustering in these areas. (Adapted with permission from the NSW Department of Industry.)
Clinical and environmental isolates alongside the type strains L. pneumophila ATCC 33152 (Philadelphia; GenBank accession no. AE017354) and L. pneumophila ATCC 33215 (Chicago 2; BioProject accession no. PRJNA239272) were grown in the laboratory, DNA was extracted, and sequencing libraries were prepared alongside all the other isolates as controls of assembly- and bioinformatics-based pipelines. In addition, two Lp1 clinical isolates from previous unrelated outbreaks in Sydney (Reference 2013 and Reference 2015) were included as unrelated outgroups.
DNA extraction and WGS.
Genomic DNA was extracted from pure cultures using the DNeasy blood and tissue kit (Qiagen, Chadstone, Victoria, Australia), with a 3-h proteinase K digestion at 56°C. The quality of each DNA sample was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific), and the 260/280 nm and 260/230 nm ratios were inspected. DNA purity was determined to be satisfactory if the 260/280 ratio fell between 1.6 and 2.2 and the 260/230 ratio fell between 1.8 and 2.2. Paired-end indexed libraries of 150 bp in length were prepared from an input 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 NextSeq 500 (Illumina).
Genome assembly and analysis.
FASTQ files were imported into Geneious (version 8.0.4) and mapped to a curated reference of L. pneumophila Philadelphia (GenBank accession no. NC_002942) using the bwa plugin (version 0.7.10) with bacteriophage, insertion sequences, and other repeat regions removed, 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 strain Lorraine (BioProject accession no. PRJNA67921). Quality-based variant detection was performed using CLC Genomics Workbench version 7.0 (CLC bio, Aarhus, Denmark). Variant detection thresholds were set for a minimum coverage of 10-fold and minimum variant frequency of 75%. SNPs were excluded if they were in regions with a minimum fold coverage of <10-fold, within 10 bp of another SNP, or within <15 bp from the end of a contig. Maximum likelihood phylogenetic trees were constructed from SNP matrices using 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 and obtaining locus numbers (14). In addition, core-genome multilocus sequence typing (cgMLST) was conducted using the Ridom SeqSphere software (Qiagen) employing gene definitions determined from closed or complete L. pneumophila genomes from NCBI GenBank (35). Using these criteria, the core genome was determined to be 1,530 genes, with an accessory genome of 1,370 genes. Further pangenome assessment and visualization were performed using the Roary pipeline (36) that includes alignment using MAFFT (37) and tree building with FastTree (38). Specific protein and nucleotide comparisons were made (including the unique T4ASS) to all available Legionella genomes on NCBI using the 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 two recently sequenced (and closed) genomes, those of LPE509 (GenBank accession no. NC_020521.1) and C1-S (GenBank accession no. CP015932.1). L. pneumophila strain Philadelphia was also included in these comparisons, given that it also contains a T4ASS that is of similar size and has the same insertion site as the T4ASS described in this study. Pairwise genome comparisons of the unique T4ASS were performed using BLASTn and visualized using Artemis (39), Easyfig (41), and Phandango (40). The presence of all characterized 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 three clinical strains (two of which were ST211). The genomes of L. pneumophila strain Paris and L. pneumophila strain 130b (GenBank accession no. NZ_CAFM01000092) were also included to enable a comparison with all 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.
Supplementary Material
ACKNOWLEDGMENTS
We thank public health professionals and epidemiologists from NSW Health and the City of Sydney, as well as laboratory scientists from NSW Health Pathology for providing clinical and environmental isolates and their assistance in collecting epidemiological and environmental information.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02020-17.
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