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Researchers Turn to NGS to Understand Growing Threat of Antibiotic Resistance in Pathogens

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NEW YORK (GenomeWeb) – Antibiotic resistance is a growing public health concern. According to the US Centers for Disease Control and Prevention, every year 2 million people acquire bacterial infections that are resistant to at least one antibiotic and 23,000 people die as a result of those infections.

Over the past four years, next-generation sequencing has become an increasingly important tool for identifying antimicrobial resistance genes and understanding how such genes are acquired over time. In some cases it has even been a useful tool for piecing together the transmission events during outbreaks.

Recently, researchers have published a number of studies illustrating how sequencing can identify resistance genes and piece together the evolution of a pathogen's genome — highlighting how NGS could potentially be used in surveillance and making clinical management decisions. One group is even developing a search engine pipeline to identify antimicrobial resistance genes from metagenomic samples.

Nonetheless, aside from a few efforts such as Europe's PATHSEEK consortium — aUniversity College London-led project to develop a sequencing platform for use in diagnostic microbiology — most of these initiatives are not large or international, which many say will be necessary to address the problem.

"Pathogen sequencing needs to have a larger role in the clinic in order for us to better understand transmission and dissemination of outbreak strains and to quickly characterize the drug resistance potential of a pathogen," Jolene Bowers, a research associate at the Translational Genomics Research Institute, told GenomeWeb.

Bowers co-led a study in collaboration with the CDC on multidrug-resistant Klebsiella pneumoniae that produce the carbapenemase enzyme, which confers resistance to the antibiotic carbapenem. The dissemination of these so-called KPC-producing organisms is largely attributed to the rise of a single strain, known as ST258. The TGen and CDC group focused on the genomic analysis of isolates from this strain and its clonal group CG258, sequencing 167 different isolates from 20 countries.

The group published its results recently in PLOS One, identifying potential genomic contributors to ST258's pathogenic success and unique genomic markers. The genomic information also helped to understand how the strain has spread globally.

The group found that ST258 isolates came from a common ancestor and acquired the carbepenem resistance gene before dissemination. Despite sequencing strains that had been collected over 17 years, the researchers found that the genomes were highly clonal. In addition, only a few mutations set ST258 apart from its clonal group, including one in the global transcription regulator.

The rapid spread of ST258 indicates "some sort of advantage over other strains," Bowers said. She said it could be to a combination of factors that created a "perfect storm." For instance, one of ST258's first emergences was in the New York City area, a major hub of global travel. In addition, its emergence also coincided with the first descriptions of the KPC gene for multidrug resistance, she said.

Brandi Limbago, a senior author on the study and deputy director of the CDC's Division of Healthcare and Quality Promotion's Clinical and Environmental Microbiology Branch, said the CDC was especially interested in studying this strain when the KPC-producing gene was discovered and it became clear that the ST258 strain was the most common one to have the gene. The CDC wanted to know "why these things co-emerged," and whether the KPC-producing strain was common only in the US or elsewhere, as well.

The CDC lists carbapenem-producing Enterobacteriaceae (a class of bacteria that includes Klebsiella) as an "urgent threat" to public health. The ST258 strain of Klebsiella pneumoniae is the most common one.

When the researchers first started the study, not much was known about ST258. In addition, it was one of the only strains known to have the KPC gene. Although it is still the most common pathogen with that gene, "we are absolutely seeing the emergence of KPC in other pathogens," Limbago said, including Enterobacter, Escherichia coli, and others. "There definitely is this possibility and actuality of the KPC gene spreading and emerging in other types of bacteria," she added, which typically occurs through mobile elements and plasmids.

Piecing together how the plasmids and mobile elements move is where sequencing can really play a role. "That piece of the puzzle is really hard to understand without sequencing," Limbago said. In the PLOS One study, for instance, the researchers were able to pick apart how the vertical transfer of the mobile element that carries the KPC gene played a role in the gene's dissemination. In addition, independent acquisition of the mobile element indicates "persistent selective pressure" on the strain to harbor the KPC gene or other genes on the same mobile element.

Limbago added that understanding these selective pressures and how mobile elements and plasmids are transferred can help in surveillance in terms of figuring out "what kinds of resistance genes are moving around and how, and what organisms [and geographies] they're moving into," she said.

Similar efforts to understand the genomics of antibiotic resistance in pathogens are being pursued elsewhere. Niyaz Ahmed, who heads the department of biotechnology and bioinformatics at the University of Hyderabad in India, told GenomeWeb that the problem is especially pressing in India because it lacks an effective antibiotic policy. Drug resistance is "often blamed on non-compliance from patients due to low education levels, a large number of non-medical entities prescribing antibiotics, over-the-counter sale of drugs, and widespread use of antimicrobial compounds in animal feed," he said.

Ahmed's group recently published a study in Antimicrobial Agents and Chemotherapy analyzing the genomes and functional attributes of subclones of the E. coli ST131 H30-Rx strain, which is associated with multi-drug resistance. The researchers compared the H30-Rx clone to another ST131 strain called SE15.

Ahmed said his lab focuses on analyzing predominantly E. coli and Helicobacter pylori genomes. In the study, the group performed whole-genome sequencing, comparative analysis, and phenotypic virulence assays, and also profiled the antibacterial responses of cells when infected with the E. coli subclones.

They found seven phage-specific regions associated with H30-Rx, but not SE15. However, within the two H30-Rx subclones, one was more virulent and had more robust antimicrobial activity and invasion ability.

Ahmed said that the study "reflects on the reality that multi-drug resistant clones of pathogens are gaining a foothold in clinical settings," and highlights the importance of using genomics and biology to "understand the mechanisms of gaining fitness advantage."

Although, he said this is a research study so could not directly inform clinical management decisions, it and others like it do provide meaningful insights that could enable the rapid identification of resistance mechanisms and prediction of a "treatment outcome with existing or modified regimens."

In addition, he said, such studies could also inform the development of an effective antibiotic policy. Future research will likely help elucidate mechanism of genetic exchange among pathogens and between pathogens and their environment to help guide policy. Aside from antibiotic use, he said, such studies could inform policy related to sanitation.

Ahmed added that "genome sequencing is the first straightforward thing one should do for an isolate as soon as it is cultured and appears to be interesting in terms of its health implications." However, he noted that in community settings and public health laboratories, whole-genome sequencing is still "technically and economically demanding," particularly in resource-limited countries.

While groups like Ahmed's and the CDC/TGen team are using sequencing to understand the evolution of pathogens and how they acquire resistance genes, another group is trying to use this collective information on drug resistance genes to create a pipeline that can be applied to metagenomic samples for rapid identification.

Dubbed Search Engine for Antimicrobial Resistance (SEAR), the pipeline and web interface identifies resistance genes directly from raw sequence data. Will Rowe, lead author of the PLOS One study that describes the pipeline, told GenomeWeb that the tool identifies known resistance genes from reference databases. He said he began designing it after unsuccessfully trying to look for resistance genes in metagenomic samples.

"You can find the pathogens and you can find the genes, but correlating them is tricky," he said. In the study, his group demonstrated the tool on two novel environmental metagenomes, 32 human fecal microbiome datasets and 126 clinical isolates of Shigella sonnei.

Rowe said that although the study is a proof of concept, he plans to continue to develop the pipeline.

While antimicrobial-resistant bacteria are a growing concern, the CDC's Limbago stressed that, at least currently, the "nightmare bacteria"— those with the KPC genes — are not yet very common. "Now is the time to control the spread," she said.

While sequencing will definitely play a role in understanding the genomes of these pathogens and how they acquire the drug resistance genes, she still thinks it is too early for it to be used clinically to make decisions on how infected patients should be treated.

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