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Team Uses Genome Sequencing To Track Bacterial Pathogen Evolution During Outbreak

NEW YORK (GenomeWeb News) – In a study appearing online yesterday in Nature Genetics, a Massachusetts-led research group described some of the genetic changes that occurred in Burkholderia dolosa bacteria over several years in individuals with cystic fibrosis who became infected with the pathogen during an outbreak in Boston.

"Tracking the genomic evolution of bacterial pathogens during the infection of their human hosts provides a direct method for observing evolutionary mechanisms in vivo and allows the identification of genes central to pathogenesis," the study authors wrote. "This study, which harnesses the combination of high-throughput sequencing and parallel evolution in the clinical setting, is a step toward a comprehensive understanding of genetic adaptation during pathogenesis."

The team, led by Alexander McAdam, director of the Children's Hospital Boston's infectious diseases diagnostic lab, Gregory Priebe, a microbiologist with Brigham and Women's Hospital's Channing Laboratory who also specializes in infectious disease and pediatrics at the Children's Hospital Boston, and Harvard Medical School systems biology researcher Roy Kishony, used genome sequencing to characterize more than 100 B. dolosa isolates collected from 14 individuals with CF over 16 years.

In the process, they found evidence of parallel evolution in isolates from different individuals involved in the outbreak, with 17 of the same B. dolosa genes showing evidence of positive selection in more than one of the patients tested.

Individuals with CF are particularly prone to long-lasting lung infections, the study authors noted, and even relatively rare pathogens can quickly spread between CF patients in a clinical setting.

In an effort to understand the influence that the human immune system has on the evolution of these pathogens over time, researchers used whole-genome sequencing to characterize pathogens from a Children's Hospital Boston outbreak involving the rare pathogen B.dolosa. The outbreak began in Boston in the 1990s, eventually infecting 39 individuals.

Using the Illumina GAIIx, the researchers sequenced the genomes of 112 B. dolosa isolates collected from 14 CF patients to a mean depth of 37 times. The isolates were resistant to many or all of the antibiotics typically used to treat infections in individuals with CF.

"This collection covers the epidemic with high temporal resolution and enables the study of parallel evolution of the same strain in multiple individuals," McAdam, Priebe, Kishony, and co-authors wrote.

After aligning sequences to a B. dolosa reference genome, the team then assessed the genetic patterns in the isolates from each individual over time, looking for genes that appeared to be under selective pressure in the human hosts.

In their initial analyses, the researchers did not find evidence of selection in coding regions of the genome based on genome-wide non-synonymous to synonymous mutation ratios.

But when they focused in on genes containing multiple mutations in several study participants, they found 17 bacterial genes that appeared to be under positive selection, including genes involved in everything from antibiotic resistance to bacterial outer membrane synthesis.

"These data told us what the pathogen experiences as its main challenges," Kishony said in a statement.

The search also led to genes involved in oxygen-sensing and oxygen-related gene regulation — including a homolog of the Escherichia coli histidine kinase gene FixL — offering potential clues to the genetic changes that B. dolosa undergoes while adapting to lower than usual oxygen concentrations in the lungs of those with CF.

"This fits well with prior studies showing that bacteria in the CF lungs are exposed to low oxygen levels," Priebe told GenomeWeb Daily News in an e-mail message. "Thus, FixL is a potential new therapeutic target."

Through phylogenetic analyses, meanwhile, the researchers were able to discern the relationships between the B. dolosa isolates evaluated in the study, allowing them to track transmission patterns between some individuals involved in the outbreak.

Still, they cautioned, because the study did not include data for all 39 individuals involved, "we cannot determine whether transmission occurred directly from one subject to another or indirectly through an individual not in our study, via a healthcare worker or through a medical device."

"Nevertheless," the study authors added, "this analysis shows that this specific epidemic was transmitted through several people during its spread, indicating the strength of this approach for the identification of the infection network of an epidemic."

Moreover, Priebe noted, the same approach should be useful for characterizing bacterial strains behind other CF infection outbreaks, such as Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus.

In the future, the team plans to look in more detail at how B. dolosa and other pathogens that infect CF patients evolve within different areas of the lung in the same infected individual.

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