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Sequencing Uncovers Antibiotic Resistance-Related Mutations in Superbug

By Andrea Anderson

NEW YORK (GenomeWeb News) – By sequencing the genomes of bacteria from two clinical samples from the same patient, researchers have identified mutations conferring resistance to the antibiotic daptomycin in an Enterococcus faecalis that was already resistant to vancomycin.

The American and Columbian team used whole-genome sequencing and other methods to characterize a pair of vancomycin-resistant enterococci isolates from a patient who died of bacteremia in a hospital in Indiana several years ago. The first was collected before treatment with daptomycin, and the second isolate was collected after. The findings, published online last night in the New England Journal of Medicine, showed that mutations in two genes are enough to produce robust daptomycin resistance in E. faecalis.

"When we compared all the genes of the whole genome, we were able to identify four mutations in four genes," first author Cesar Arias, an infectious disease researcher and director of the Laboratory for Antimicrobial Research at the University of Texas Medical School at Houston, told GenomeWeb Daily News. "And two of them, we were able to demonstrate, were directly related with the development of this [daptomycin-] resistant phenotype."

Some of the same genes and pathways were altered in post-daptomycin treatment clinical samples from a patient infected with another vancomycin-resistant species called E. faecium, suggesting the same pathways may contribute to daptomycin resistance in other difficult to treat enterococcal species.

"These bacteria, vancomycin-resistant enterococci, are particularly difficult to treat," Arias said. As a group, the bacteria are also among the second most common type of "superbug" isolated in US hospitals.

The vancomycin-resistant enterococci are especially dangerous in individuals with compromised immunity, he noted, and can cause bloodstream, urinary tract, and heart valve infections, as well as infections associated with catheters.

Two antibiotics, linezolid and quinupristin-dalfopristin, are approved by the US Food and Drug Administration for treating enterococcal infections resistant to first-line antibiotics such as vancomycin, the researchers noted. But treatment with these antibiotics can be challenging and is not always effective.

"These antibiotics have limited spectra, you have a lot of toxicities, and the outcomes are really not the best," Arias said.

Consequently, physicians frequently turn to off-label use of another drug: the lipopeptide antibiotic daptomycin, which exerts an effect on enterococci by targeting the bacteria's cell membrane.

"Daptomycin is practically the only drug that has in vitro activity that is very good against these bugs," Arias said. "It really kills bacteria very nicely in the test tube."

Even so, he explained, daptomycin is not as potent in patients as it is in vitro when used at standard doses. And antibiotic resistance can be a problem for daptomycin too, though the strategies that enterococci use to dodge the drug are poorly understood.

"The development of resistance to this drug is a very bad thing, because we are left with nothing to treat these particular organisms," Arias said. "It becomes crucial to understand how to use it better and, of course, how the bug manages to resist its action."

To look for potential resistance mechanisms, study authors from the Washington University Genome Institute used the Illumina GAIIx platform to do paired-end sequencing of a pre-daptomycin treatment isolate of E. faecalis called S613 and a post-daptomycin isolate (R712), both from the same patient.

"We were able to acquire these isolates and sequence the genomes before and after, when the bug was susceptible and when the bug was resistant," Arias explained. "The power of these findings lies in the fact that it was from a real patient, a real clinical situation."

When the team compared the genome of the daptomycin-resistant isolate R712 with the genome of the susceptible S613 isolate, they found potential daptomycin-resistance related mutations affecting four genes.

In particular, the vancomycin-resistant isolate that had survived daptomycin treatment had in-frame deletions in three genes not altered in the pre-daptomycin isolate: the phospholipid metabolism genes gdpD and cls, and a predicted membrane protein-coding gene, liaF.

Mutations in the gdpD and liaF genes also turned up in an S613-derived isolate that had been coaxed into becoming daptomycin-resistant over a few weeks via exposure to increasing antibiotic concentrations, suggesting that that bug used similar pathways to reach daptomycin resistance in the lab.

Changes to the liaF gene seem to initiate some early stages of this resistance, the researchers found, since swapping out the version of the gene found in the daptomycin-sensitive patient isolate for the one in the resistant R712 isolate produced enterococci capable of growing in higher daptomycin concentrations.

Trading the gdpD allele alone did not alter the daptomycin resistance of the pre-daptomycin isolate. But when the team took pre-daptomycin isolates and replaced both the gdpD and liaF genes with versions found in the resistant R712 isolate, they produced bugs with daptomcyin resistance levels comparable to those found in R712 itself.

Meanwhile, when they looked at pre- and post-daptomycin treated isolates from an individual infected with another vancomycin-resistant enterococcal species, E. faecium, the team detected daptomycin resistance-related mutations in cls, liaF, and other genes participating in a so-called LiaFSR regulatory system with liaF.

"This supports that these genes are very important for the development of this resistance during therapy," Arias said. "The mutations may change, but the end effect may be the same."

Based on findings from their genetic analyses and from transmission electron microscopic examinations of daptomycin-susceptible and -resistant cells, researchers speculated that resistance-related mutations somehow distort the bacterial cell envelope, interfering with the drug's activity.

It's possible that changes to other genes could confer daptomycin resistance as well, Arias noted, though additional studies are needed to determine whether that's the case. "There's always the possibility that other things are going on," he said, "and it may vary depending on what biological situation this organism is facing.

He and his colleagues plan to look for resistance mechanisms in other "superbug" strains, as well as follow-up experiments to try to understand the functional consequences of the genetic changes detected in E. faecalis.

"Once we understand how the gene changes actually produce the [antibiotic resistance] problem, we can attack that," Arias explained. "That would be the perfect kind of backbone to develop molecules to attack those targets."

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