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Genomic Analysis Reveals Burkholderia Mutations, Evolution in Cystic Fibrosis Patient

NEW YORK (GenomeWeb) – Researchers have sequenced Burkholderia samples collected from the lungs of a cystic fibrosis patient over the course of 20 years, and have found that the bacteria evolved, with some genes acquiring multiple mutations.

The Universidade de Lisboa-led team sequenced and analyzed 22 Burkholderia multivorans samples obtained from a female CF patient and found that it developed into four clades. Some 10 genes— mostly involved in gene expression regulation and lipid metabolism — acquired three or more mutations, which suggests they might be targets of selection, as Lisboa's Leonilde Moreira and her colleagues reported in mSystems today.

"These mutations corresponded to what was happening physically with the patient, so we could see that those mutations were not just random — they were specific targets that affected the physiology of the bacteria," Moreira said in a statement.

She and her colleagues sequenced the genomes of 22 B. mulitovorans isolates from a CF patient in Vancouver between 1993 and 2013. Before 1993, the patient was infected by Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, and was co-infected with those and B. multivorans between 1993 and 1996. She was hospitalized once because of a CF-related illness, but had been treated multiple times with antibiotics.

By mapping the various B. mulitovorans isolates' genomes against that of the first isolate, the researchers uncovered 399 SNPs and 88 indels. The last isolate collected accounted for 196 of these mutations, and as it harbors a frameshift mutation in its mutL mismatch repair gene, the team concluded that it has a hypermutator phenotype. Excluding that last isolate, the researchers estimated a mutation rate of 2.4 SNPs per year for the isolates.

Based on both those SNPs and indels, the researchers constructed a phylogenetic tree for B. mulitovorans from this patient. The isolates fell into four major clades, with clade C1 evolving early and another lineage then diversifying into clades C2, C3, and C4. While various lineages coexisted at any given time, the last seven isolates were all from C3, the researchers noted.

Most of the mutations they uncovered were nonsynonymous and located near coding regions, suggesting that there were periods of diversification and positive selection, followed by periods of neutral evolution, Moreira and her colleagues wrote in their paper.

The early mutations — those found in all clades or in all clades except C1 — were enriched in genes involved in transcription, signal transduction, and lipid metabolism. For instance, one of the three mutations found in all isolates was a nonsynonymous mutation in a gene encoding a FabD homolog involved in fatty acid biosynthesis.

Ten genes harbored three or more different mutations, the researchers noted, and eight of these fell in coding regions. One gene with six mutations — that evolved independently in three clades — encodes a conserved Fis family transcriptional regulator. Another gene with multiple mutations encodes a fixL homolog that's been linked with oxygen sensing in other organisms, indicating that this mutation may have helped B. mulitovorans adapt to the microaerophilic conditions of CF-affected lungs.

Other mutations may have helped B. mulitovorans combat antibiotics. The researchers reported a mutation present at the root of the C2, C3, and C4 clades in a homolog of the emrA/hlyD gene. That gene encodes a membrane fusion protein of a bacterial tripartite efflux pump, a pump that could help eliminate toxic compounds. Isolates with the mutated emrA allele, the researchers reported, had increased resistance to piperacillin plus tazobactam, as compared to earlier isolates without the mutated form.

Likewise, a mutation to the rpfR homolog gene may have enabled some isolates to produce more biofilm and better adhere to epithelial cells.

Moreira and her colleagues also correlated the changes in the bacteria to the patient's lung function. They found that a period of declining lung function in the patient between 1998 and 2006 coincided with the rise of clade C3, of isolates with increased biofilm production and antibiotic resistance phenotypes, and with a change in co-infecting pathogens.

"[T]his dynamic suggests that monitoring these evolutionary and molecular patterns could be used to design responsive therapies designed to limit population diversity and disease progression," Moreira said. "Altogether, our observations suggest that B. multivorans populations, during long-term colonization of the CF lungs, either directly or indirectly target adherence, metabolism, and changes in the cell envelope related to adaptation." She added that her team is studying B. multivorans in a further 10 CF patients.