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Regional Isolation Drives Diversification of Bacteria in Cystic Fibrosis Lungs

NEW YORK (GenomeWeb) – Isolation of bacteria within different regions of the lungs of cystic fibrosis patients drives their diversification, according to a phylogenetic study appearing in Cell Host & Microbe today.

Researchers from the University of Washington School of Medicine dissected 10 pairs of lungs from cystic fibrosis patients — obtained after lung transplantation — and examined some 12,000 isolates of Pseudomona aeruginosa inhabiting various lung regions. Phylogenetic analysis indicated that all P. aeruginosa infecting a lung were related, but it also showed that as the bacteria became isolated in different lung regions, they began to evolve differences in treatment resistance, nutritional requirements, and more.

"What made this so important to us is that the bacterial populations inhabiting different lung regions varied dramatically in terms of their antibiotic resistance and virulence," first author Peter Jorth from the University of Washington School of Medicine said in a statement. "This diversity could affect the patients' health."

Jorth and his colleagues sampled both left and right lungs from 10 cystic fibrosis patients at three sites: the upper, middle, and lower lobe airways. Using 16S rRNA sequencing, they determined the bacterial genera present in the samples, finding the vast majority to be P. aeruginosa.

P. aeruginosa isolates from different lung regions exhibited different phenotypes, including virulence and resistance phenotypes, the researchers noted. For instance, about 40 percent of the P. aeruginosa isolates they obtained from the right upper lobe of one patient were resistant to the antibiotic ciprofloxacin, while all right lower lobe isolates were sensitive to it.

These regional populations, they reported, are distinct. The researchers used phenotypic data to classify the isolates into subpopulations, and found that each patient harbored between 12 and 40 bacterial subpopulations in their lungs and that each lobar region contained between one and three of those sub-populations.

Through a proteomic analysis, the researchers found that regional bacterial populations show protein expression differences. In addition, they found that isolates found together share a greater degree of functional similarity than isolates from far-flung regions.

This, Jorth and his colleagues said, suggested to them that P. aeruginosa in the various lung regions may be evolving independently.

After sequencing the genomes of 96 isolates from the upper, middle, and lower lobes from three patients, the researchers constructed phylogenetic trees. In all three, they noted that the bacteria clustered based on the region from which they were obtained, a finding they said is characteristic of genetic compartmentalization.

Using that sequencing data, the researchers also examined the genetic diversity of the P. aeruginosa from these three patients. In patient one, they uncovered 328 SNPs, while patients two and three, whose infecting lineage exhibited a hypermutator phenotype, had a respective 3,169 and 1,653 SNPs.

For patient one, Jorth and his colleagues surveyed the functional differences between isolates from various lung regions, finding that subpopulations living in upper and lower lobes differed in their growth capacity, stress and antibiotic tolerance, and virulence.

These isolates, they found, differ by 19 SNPs. The upper lobe isolate, for instance, contains a SNP in the ciprofloxacin target topoisomerase parE and a SNP in exsD, a key negative regulator of the type 3 secretion system, which is among the most potent bacterial virulence factors.

The sequencing data noted that the exsD SNP was present in 10 out of 13 isolates from the left upper lobe, but only in three of the 47 isolates obtained elsewhere.

Based on their finding of strong signals of genetic compartmentalization, regional isolates with similar protein expression profiles, and spatially distinct distribution of bacterial phenotypes, the researchers said that their study indicates that isolation in different lung regions helped drive the diversification of P. aeruginosa in the lungs of cystic fibrosis patients.

"Even when a single strain of bacteria causes a chronic infection, evolution with human organs can produce diverse families of related bacteria," senior author Pradeep Singh from UW said in a statement. "This may be part of what makes treatment so difficult, because when bacteria sensitive to one kind of stress are killed, functionally different siblings are there to take their place."