Finding risks in a flood of genetic data

Sequencing improvements aid resistance study, outbreak response
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Faster, cheaper genome sequencing is helping public health researchers identify the risks of drug resistance and medical treatment failure.

The sequences also may help federal investigators find outbreak sources, by geographic location and species, as well as guide vaccine and antimicrobial development. Dr. Patrick McDermott, director of the National Antimicrobial Resistance Monitoring System, said the use of benchtop sequencers to read full genomes of isolates is, for data collection, like drinking from a fire hose.

"It’s going to change infectious disease medicine,” he said. “It’s going to change microbiology. It’s already doing that, in fact.”

Genotyping pathogens through pulsed-field gel electrophoresis can require days of work and hundreds of dollars, whereas commercial sequencing appliances can produce more information at costs of $40 per isolate and require less labor, he said. The types of benchtop machines used by the Food and Drug Administration  break pathogen genomes into small fragments and generate millions of overlapping sequence reads, which the sequencers align and compare against reference sequences.

Illumina MiSeq
The Illumina MiSeq, shown, is an example of the appliances used for genome sequencing, and the Food and Drug Administration included images of the device in videos on whole-genome sequencing. (Courtesy of Illumina Inc.)

The new information can help show how resistance spreads, how various alleles cause similar resistance, how common resistance-associated traits are, and, through further testing, how antimicrobial use could select for further resistance.

“We had a recent surge in gentamicin-resistant Campylobacter in NARMS,” he said. “And, when we did sequencing on them, we found just this array of genes we had never seen before, which we never would have detected if we had done the old methods.”

An August 2014 scientific article in Emerging Infectious Diseases, “Rapid whole-genome sequencing for surveillance of Salmonella enterica serovar Enteritidis,” describes a demonstration of benefits from whole-genome sequencing in outbreak surveillance. The article indicates use of a benchtop sequencer in a retrospective study of an S enterica serovar Enteritidis outbreak provided vast improvement over PFGE in detecting relationships among illnesses, as well as suggested a possible cause for the outbreak, the source of which had been unidentified.

This year, the NARMS program, a collaboration of the FDA, Centers for Disease Control and Prevention, and Department of Agriculture, published on GenBank the sequencing data for about 400 Salmonella isolates found in retail meat throughout 2014 and the first half of 2015. In an accompanying announcement from the FDA, the NARMS officials noted that the genetic data list drug resistance–associated genes and mutations.

Sequencing helped identify genes associated with resistance to quinolone antimicrobials, including a plasmid-associated gene linked with ciprofloxacin resistance and found in an isolate recovered from pork. The gene is particularly concerning because of the risk it could be transmitted to other Salmonella strains, the announcement states.

And one isolate found in chicken during 2014 and resistant to ceftriaxone had an extended-spectrum beta-lactamase gene, which confers resistance to beta-lactam antimicrobials such as third-generation cephalosporins and can reduce treatment options.

“Studies are ongoing to see if this finding points to a broader distribution of this important trait in Salmonella from other sources,” the announcement states.

The sequencing data also have revealed “the mechanisms underlying each of the resistance phenotypes observed, and how they differ by source,” the announcement states.

For example, Salmonella isolates found in chicken and ground beef tended to contain beta-lactamases associated with more extended activity than those found in turkey and pork, and they confer resistance to third-generation cephalosporins.

Additional study is needed to identify whether differences among isolates, by meat source, are stable and helpful in identifying the sources of human illnesses. Separate FDA information indicates whole-genome sequencing also can be used to evaluate whether a pathogen is persistent in a particular environment, such as Salmonella bacteria in surface water near mid-Atlantic farms where tomatoes are grown, and how bacteria benign to humans can mutate to become a pathogen.

Dr. McDermott noted that the federal government is giving the public access to its genome database, which includes antimicrobial resistance information.

“We take seriously the requirement for a global approach to the resistance problem, and we’re making our information available to anyone who wants to see it,” he said.

Brittany Behm, a spokeswoman for the CDC, provided a statement that the CDC also started performing routine whole-genome sequencing on antimicrobial-resistant nontyphoidal Salmonella isolates collected during surveillance in 2014 and all nontyphoidal Salmonella isolates collected through surveillance in 2015. That information is intended to help identify antimicrobial resistance determinants.

The CDC also has conducted targeted sequencing to characterize genes connected with drug resistance and found in NARMS-collected isolates starting in the early 2000s.

“Data provided by the NARMS program helps inform the development of public health interventions and policies designed to protect people from the threat of resistant enteric infections,” Behm said in the provided statement.

State authorities also are adopting whole-genome sequencing. The New York Department of Health’s Wadsworth Center Laboratory sequences foodborne pathogens in the state as well as others requested by the CDC or FDA. The laboratory gathers whole sequences from about 1,000 samples related to foodborne illness each year and 2,000 genetic fingerprints through PFGE each year. PFGE is used on Salmonella, Escherichia coli, and Listeria monocytogenes isolates recovered from foods and people with illnesses, according to a statement provided by the health department.

Officials with the New York department said whole-genome sequencing could link more outbreaks with their sources, decrease the time to identify those sources, and help characterize drug resistance, toxicity, and virulence.

Whole-genome sequencing also is helping NARMS gather genetic data despite a rise in use of culture-independent diagnostic tests, such as enzyme-linked immunosorbent assay, Dr. McDermott said. Because of that shift, NARMS has been having trouble finding certain pathogens for further study, such as human-source isolates of Campylobacter.

He also expects the NARMS program will further expand into meta­genomics, isolating and sequencing all DNA in complex samples to examine genes of interest, although he does not know when that could occur.

“You can figure out in a snapshot the taxonomic structure that is sampled—you know, which different families of bacteria are there—and just drill right down from family down to species down to resistance genes,” he said.