Fig 1.
Patient timeline of treatment and B. pseudomallei isolate collection.
A) CT scan shows damaged, widened airways (green circle) in the lower left lobe that required surgery on day 1134. B) Chest X-ray after lobectomy. C) Patient’s sputum in cup. D) Timeline of isolate collection. From excised lung tissue, we cultured 41 isolates on day 1134 (red circle).
Fig 2.
The location of large deletions in P314 genomes relative to MSHR1435.
Whole genome sequencing of 120 B. pseudomallei collected over 17 years reveals large chromosomal deletions across both chromosomes. The genomes’ order from the outside to inside concentric circles corresponds to the top to bottom order on the phylogenetic tree (Fig 3). This inside-to-out arrangement approximates the evolutionary trajectory of the infecting B. pseudomallei population. MSHR1435, which is used as the reference, is outermost and is a closed genome while all others are high quality drafts. All environmental isolate genomes (including MSHR1435) are basal in the phylogeny (Fig 3) and found in the outermost rings (green) of the concentric circles. Raw sequence reads were mapped to the reference chromosomes and any base with a depth of coverage of <25% of the average genome coverage was considered to be missing. The figure was constructed with Circos [43]. Coordinates for deleted regions are as follows: 1(2700183–2733804), 2a (2196965–2198380), 2b (2141162–2157867), 2c (2106537–2157867), 2d (2006273–2214755), 3 (204194–294524), 4a (1322983–1479924), 4b (1375399–1474558), 5 (1551804–1588830), 6a (1874062–1994243), 6b (1934891–1974776), 6c (1956279–2122568), 6d (2069785–2099797), 7a (2429295–2552683), 7b (2546831–2555624).
Fig 3.
(A) Phylogenetic tree showing evolutionary locations of polymorphism, (B) day of collection for each isolate, and (C) phenotypic characteristics (virulence, motility, and three colony morphotypes—W = wild type, A = small colony variants, B = fastidious). Core genome SNPs are represented as black branch segments, while non-core genome SNPs are gray. The large deletions shown in Fig 2 are green branch segments. We previously compared the genomes in blue [25]. Where mixtures are observed, the major and minor components are labeled. Methodological details for phylogenetic reconstruction are provided in the materials and methods section.
Fig 4.
Sequence read mapping to a non-ribosomal peptide synthetase (NRPS) gene from different isolates.
The Illumina reads from each of the isolates in this study were aligned to the NRPS gene from the MSHR1435 reference (MSHR1435 locus CXQ84_31440). This figure is a higher resolution representation of the read mapping shown in Fig 2 and covers the 6c and 6d regions shown there. Twelve examples of this analysis are illustrated with six environmental and six clinical isolates, with the complete isolate set present in the supplemental material (S1 Table). The read mapping shows the early loss of the internal gene sequences around position 4500 and later deletions in the 3’ region. Isolate are arranged from top to bottom in the same order as in the phylogenetic tree (Fig 3). These same isolates were all used for animal challenge experiments (see below).
Fig 5.
Convergent gene deletions among long-term carriage and non-host-adapted genomes.
Genes from the 7 deleted regions (Fig 2) found among P314 isolates are mapped against 51 B. mallei genomes, chronic B. pseudomallei genomes from 7 CF patients, and a diverse panel of acute/environmental B. pseudomallei genomes. Parts of each of the seven regions deleted among P314 genomes were also deleted in B. mallei genomes. Portions of three of the seven regions were also deleted in some genomes associated with long-term CF infections, however two of these deletions were evident in both the initial and later isolates, suggesting that their absence is not necessarily linked to host adaptation. Indeed, these genes are also absent in the diverse panel of non-host-adapted (acute and environmental) genomes. Conversely, part of region 4b was deleted over the course of a CF infection and among all B. mallei. None of the genes from this region were deleted in any of the genomes from the acute and environmental panel, suggesting that their deletion may be a specific adaptation to long-term carriage. Deletions in the 6d region correspond to the NRPS gene (Fig 4) and are also observed in other genomes from these panels. Putative deletions seen among P314 genomes were assumed to be due to fragmented assemblies if they could not be verified with read mapping (Fig 2) and were thus not assigned a number/letter.
Fig 6.
Mortality and morbidity of P314 isolates and P314-associated environmental isolates.
A-C) BALB/c mice were intranasally inoculated with B. pseudomallei isolates (n = 5/group). Mice were monitored for clinical score and were weighed daily for weight loss and if needed were euthanized (A and B). The inoculum for the single dose (A) and the 1435 decreasing dose (B) was back titrated and the CFU/mouse was determined and listed in the figure or below. Back titration results: (A) P314-associated environmental isolates, 1435–2,600 CFU, 1436–3,400 CFU, 1450–2,800 CFU, 1498–3,025 CFU, 1887–6,000 CFU, 1895–6,250 CFU. P314 isolates, 1070–9,250 CFU, 1288–9,550 CFU, 1043–11,040 CFU, 1046–10,350 CFU, 1218–10,150 CFU, 1285–10,450 CFU. Survival significance was determined by Mantel-Cox log rank analysis comparing the individual environmental isolates to the patient isolates. C) Individual percent change in mouse weights at day 2 are shown by dots (n = 5/group) and the mean weight is shown by the bar. The isolates are also listed by the relatedness to the environmental isolates cluster (right to left). The change in weight significance was determined with a Kruskal-Wallis test by comparing the change in weight at day 2 for each isolate listed. BALB/c mice were also challenged with 1655 with no mortality but the experiment was terminated early at 14 days (1655) and is not shown. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.
Fig 7.
Tree showing location of lobectomy samples suggest one non-segregated population. Samples from the same region are found across clades on the tree and multiple clades each contain samples from multiple sites.
Fig 8.
Size of pan (black) and core (red) genome with increased random sampling of genomes show a closed genome and the reduced size of some genomes due to genomic deletions.
Fig 9.
Population diversity dynamics.
Estimates of diversity (Faith’s PD and Observed OTUs) in each time period and accumulating over time. This shows that mutations in the first part of year 1 cause a rapid increase in accumulated diversity that continues with time. Continued extinction of earlier genotypes suppresses this accumulation of diversity at each time period. This changes in year 4 when diversity in this time period dramatically increases and, despite the extinction of most newly emerged lineages, is maintained thereafter due to the persistence and continued divergence of two clades. The number of observed OTUs and isolates in each time period remains mostly constant with the exception of year 4 due to the large number of isolates collected from the lobectomy.
Fig 10.
Bayesian estimation of infection date and population dynamics.
SNPs from 120 clinical genomes longitudinally sampled over 17 years, were evaluated to determine the estimated time of infection (TMRCA for all clinical isolates) and population dynamics over the course of the infection. The 95% highest posterior density interval is in green and suggests a median time of infection of 343 (mean: 414, 95%HPD: 10–999 days) days prior to the first hospital visit. Posterior probabilities that are greater than 0.80 are indicated for all interior branches. The Bayesian Skygrid (top, purple) demonstrates the fluctuation of the effective population size over the course of the infection. Three important events are indicated: 1) Time of Infection, 2) Time that the first isolate was collected, and 3) Time when lung lobectomy was performed. Iterative downsampling of isolates at the time of the lobectomy show that effective population size estimates are not driven by the numerous samples collected at the time of the lobectomy (S5 Fig).