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Within-Host Evolution of Burkholderia pseudomallei during Chronic Infection of Seven Australasian Cystic Fibrosis Patients.

ABSTRACT: Cystic fibrosis (CF) is a genetic disorder characterized by progressive lung function decline. CF patients are at an increased risk of respiratory infections, including those by the environmental bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Here, we compared the genomes of B. pseudomallei isolates collected between ~4 and 55 months apart from seven chronically infected CF patients. Overall, the B. pseudomallei strains showed evolutionary patterns similar to those of other chronic infections, including emergence of antibiotic resistance, genome reduction, and deleterious mutations in genes involved in virulence, metabolism, environmental survival, and cell wall components. We documented the first reported B. pseudomallei hypermutators, which were likely caused by defective MutS. Further, our study identified both known and novel molecular mechanisms conferring resistance to three of the five clinically important antibiotics for melioidosis treatment. Our report highlights the exquisite adaptability of microorganisms to long-term persistence in their environment and the ongoing challenges of antibiotic treatment in eradicating pathogens in the CF lung. Convergent evolution with other CF pathogens hints at a degree of predictability in bacterial evolution in the CF lung and potential targeted eradication of chronic CF infections in the future.IMPORTANCEBurkholderia pseudomallei, the causative agent of melioidosis, is an environmental opportunistic bacterium that typically infects immunocompromised people and those with certain risk factors such as cystic fibrosis (CF). Patients with CF tend to develop chronic melioidosis infections, for reasons that are not well understood. This report is the first to describe B. pseudomallei evolution within the CF lung during chronic infection. We show that the pathways by which B. pseudomallei adapts to the CF lung are similar to those seen in better-studied CF pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia complex species. Adaptations include the accumulation of antibiotic resistance, loss of nonessential genes, metabolic alterations, and virulence factor attenuation. Known and novel mechanisms of resistance to three of the five antibiotics used in melioidosis treatment were identified. Similar pathways of evolution in CF pathogens, including B. pseudomallei, provide exciting avenues for more-targeted treatment of chronic, recalcitrant infections.

SUBMITTER: Viberg LT

PROVIDER: S-EPMC5388805 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Within-Host Evolution of Burkholderia pseudomallei during Chronic Infection of Seven Australasian Cystic Fibrosis Patients.

Viberg Linda T LT Sarovich Derek S DS Kidd Timothy J TJ Geake James B JB Bell Scott C SC Currie Bart J BJ Price Erin P EP

mBio 20170411 2

Cystic fibrosis (CF) is a genetic disorder characterized by progressive lung function decline. CF patients are at an increased risk of respiratory infections, including those by the environmental bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Here, we compared the genomes of B. pseudomallei isolates collected between ~4 and 55 months apart from seven chronically infected CF patients. Overall, the B. pseudomallei strains showed evolutionary patterns ...[more]

PMID: 28400528

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Project description:UNLABELLED:Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host. IMPORTANCE:Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Project description:Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Project description:Burkholderia multivorans is an opportunistic pathogen capable of causing severe disease in patients with cystic fibrosis (CF). Patients may be chronically infected for years, during which the bacterial population evolves in response to unknown forces. Here we analyze the genomic and functional evolution of a B. multivorans infection that was sequentially sampled from a CF patient over 20 years. The population diversified into at least four primary, coexisting clades with distinct evolutionary dynamics. The average substitution rate was only 2.4 mutations/year, but notably, some lineages evolved more slowly, whereas one diversified more rapidly by mostly nonsynonymous mutations. Ten loci, mostly involved in gene expression regulation and lipid metabolism, acquired three or more independent mutations and define likely targets of selection. Further, a broad range of phenotypes changed in association with the evolved mutations; they included antimicrobial resistance, biofilm regulation, and the presentation of lipopolysaccharide O-antigen repeats, which was directly caused by evolved mutations. Additionally, early isolates acquired mutations in genes involved in cyclic di-GMP (c-di-GMP) metabolism that associated with increased c-di-GMP intracellular levels. Accordingly, these isolates showed lower motility and increased biofilm formation and adhesion to CFBE41o- epithelial cells than the initial isolate, and each of these phenotypes is an important trait for bacterial persistence. The timing of the emergence of this clade of more adherent genotypes correlated with the period of greatest decline in the patient's lung function. All together, our observations suggest that selection on B. multivorans populations during long-term colonization of CF patient lungs either directly or indirectly targets adherence, metabolism, and changes in the cell envelope related to adaptation to the biofilm lifestyle. IMPORTANCE Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient's lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression.

Dataset Information

Within-Host Evolution of Burkholderia pseudomallei during Chronic Infection of Seven Australasian Cystic Fibrosis Patients.

Publications

Within-Host Evolution of <i>Burkholderia pseudomallei</i> during Chronic Infection of Seven Australasian Cystic Fibrosis Patients.

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