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JCVI Bacterial Sequencing Interrogates Bleeding Edge of Antibiotic Resistance Genes


NEW YORK (GenomeWeb) – More than a year into a $25 million grant from the National Institute of Allergy and Infectious Diseases, the J. Craig Venter Institute is trying to sequence bacteria that are the cause of great concern due to their growing antibiotic resistance.

JCVI is in the second year of funding of the five-year grant. Of the $5 million awarded in 2015, $310,095 will go towards sequencing several gram-negative bacteria that most worry scientists and clinicians. Klebsiella pneumoniae and its gene conferring resistance to carbapenems, a class of antibiotics, are particularly concerning. The grant will study how the K. pneumoniae carbapenemase (KPC) gene is spreading, especially to two other types of bacteria.

With a focus on basic science, JCVI Scientific Director Mark Adams said the institute's Genome Center for Infectious Disease is looking to find not only the genes driving antibiotic resistance but also how and why they've spread so far throughout bacterial communities. The sequencing might be able to better inform molecular diagnostic testing for antibiotic resistance.

Answering these questions will require whole-genome sequencing, Adams told GenomeWeb. PCR testing can only find the specific resistance genes, but in the cases studied under the grant, it appears that will not be enough. Genes are one thing, but their context in the genome that drives gene expression could prove even more important.

With the grant money, Adams and JCVI are focusing on two types of bacteria in addition to K. pneumoniae, a genus known as Enterobacter and Acinetobacter baumannii, a ubiquitous bacteria found in soils the world over.

"One of the complications is that the species relationships are not very well resolved right now," in Enterobacter, Adams said. "We just call it Enterobacter and cast a wide net for it." Nonetheless, the KPC gene is rapidly spreading into those organisms.

For Adams, A. baumannii is somewhat of a pet organism. He has worked with it in the past and said he currently has a separate grant to also study it further. It's a growing threat to human health and appears to have several traits that could help explain how antibiotic resistance is acquired or developed.

"It's a very dynamic organism," Adams said about A. baumannii. In the past, the bacterium wasn't a serious health concern because it was readily treatable, but that's often no longer the case. "In the last 10 years, now more than two thirds of isolates are multi-drug resistant, resistant to at least three different classes of drugs," Adams said. "There's quite a bit of genetic exchange. Insertion sequences are popping around and that's changing the resistance profile even among isolates that are very closely related in time and space."

Through a collaboration with Robert Bonomo of Case Western Reserve University and Cleveland VA Medical Center, Adams has access to a collection of A. baumannii samples from infected patients going back almost a decade. "That's a tremendous resource for studying how the bacterial population has changed," Adams said.

By sequencing all those samples, Adams hopes to elucidate some quirky features of A. baumannii's resistance mechanisms.

Beta-lactamases are a critical component of resistance, enzymes that inactivate many standard drugs including penicillin and cephalosporin. There are several genes that encode beta-lactamase proteins in bacteria; while one is probably enough to confer resistance, Adams said that A. baumannii appear to stockpile them. "They're not happy with just one mechanism for defeating the antibiotics; they like to keep adding them on. We'll often often find isolates with several beta-lactamases or several contributors to [ciprofloxacin] resistance," he said.

While stockpiled and readily transmissible antibiotic resistance genes are worrisome, they don't necessarily reveal the whole picture. In the case of A. baumannii, just because a microbe has a gene for antibiotic resistance, doesn't mean it's actually resistant to treatment with that drug. Genes, after all, still need to be expressed and the ability to express those antibiotic resistance genes varies wildly within the species.

Almost every strain of A. baumannii has a gene for a carbapenemase enzyme that confers resistance to the drugs imipenem and meropenem, Adams said, but it's not expressed at a very high level in most of them, making them susceptible. "But if an insertion sequence pops in, in front of gene, a promoter in the insertion sequence can cause overexpression of the gene and resistance to those drugs," he said.

Testing for the presence of the gene will almost always come back positive, but the result doesn't say anything about the bacteria's ability to actually resist treatment. "Knowing the genetic context is what's important," Adams said. More information about genomic context could improve molecular diagnostic testing by pointing out instances where the presence of a gene indicates a resistant phenotype but the context actually suggests it's susceptible to a drug.

Another contextual setting that Adams wants to study is the relation between epigenetic marks on DNA and gene expression. In bacteria, methyltransferase adds a mark on DNA at special sites that can protect the DNA from cleavage by restriction enzymes.

"We can infer the DNA base modification patterns that are present in the DNA if we sequence at adequate depth," Adams said, noting that JCVI researchers are using sequencers from Pacific Biosciences. "Evidence seems to be increasing that these methylation patterns can affect gene expression in eukaryotes."

While he admits that methylation in prokaryotic species probably doesn't work the same way, even if there are correlations with gene expression, it's worth looking in to. "We're trying to study whether we can find conditions where that's true, especially related to antibiotic exposure; looking at gene expression is obviously an important a part of that," he said.

Adams said base modification is a "wildly understudied" part of bacterial physiology, in part because the PacBio single molecule sequencing platform needed to look at the methylation signature is expensive.

"The polymerase as its replicating across a methylated base has a slightly different incorporation time for the nucleotide," Adams said. The software running on the PacBio sequencer is able to take advantage of that to recognize modified bases. Illumina sequencing, which is prevalent at JCVI, isn't able to account for modified bases.

Another advantage of PacBio's technology is in sequencing of plasmids, which often carry resistance genes between species. "Plasmids in particular don't assemble well with standard Illumina strategies, yet they're a very interesting part of the resistance repertoire," Adams said.

Adams pointed to a recently published paper concerning resistance to colistin, a last-line antibiotic, that he said shows the value of looking at gene expression in resistance. "Gene expression analysis helped show a new mechanism of resistance that hadn't been appreciated before in a regulatory system," Adams said.

Antibiotic resistance, especially in A. baumannii and Enterobacter, is a growing concern — and soon community acquired infections, such as those in the urinary tract, could become treatable only with intravenous antibiotics. "That would change the nature of that as a health concern," Adams said, noting that there aren't new drugs coming out for gram-negative bacteria like A. baumanii and Enterobacter.