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Team Takes GWAS Approach to Characterize Antibiotic Resistance in Opportunistic Pathogen

NEW YORK (GenomeWeb) – In a study appearing online last night in PLOS Genetics, researchers from the Wellcome Trust Sanger Institute and elsewhere outlined the genome-wide association study approach they used to track down variants involved in antibiotic resistance in the opportunistic pathogen Streptococcus pneumoniae.

"Uncovering all the single-letter differences underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology," the Sanger's Julian Parkhill, co-senior author on the study, said in a statement. "GWAS will enable this by allowing us to pinpoint the location of the real genetic culprits rather than our current mapping that gets us only to the right general area."

To account for the large sets of linked variants, or haplotype blocks, that tend to occur in the genomes of S. pneumoniae and other bacteria, he and his team began by comparing sets of S. pneumoniae isolates from the US and Thailand that were sensitive or resistant to beta-lactam type antibiotics such as penicillin, including almost 3,000 isolates collected from patients treated at a refugee center on the Myanmar-Thailand border.

By folding bacterial recombination patterns into their GWAS, the study's authors were better able to accurately narrow in on variants with apparent ties to antibiotic resistance in each S. pneumoniae population, rather than being stumped by haplotype blocks containing multiple variants.

So far, the search has uncovered just over 300 SNPs at 51 sites in the genome that correspond with beta-lactam resistance in bugs from lineages that are and are not targeted by existing vaccines.

"The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs," Parkhill and his colleagues wrote.

"While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics," they added.

Although S. pneumoniae is commonly found in the nose and throat of healthy individuals, it can cause serious infections ranging from pneumonia to meningitis and has been implicated in an estimated 1.6 million deaths around the world each year.

Such infections can be tricky to treat owing to growing resistance to beta-lactam and other antibiotics, the researchers explained, prompting interest in understanding the genetic roots of this resistance.

To that end, the team pulled together more than 3,700 S. pneumoniae isolates, including 3,085 from Thailand and 616 from the US. The large sample population was designed to deal with the relative dearth of recombination events in bacterial populations, which can mask causal variants amongst innocuous SNPs in linkage disequilibrium with them.

Starting from genome sequences for the isolates that had been generated using Illumina HiSeq 2000 instruments, the researchers searched for SNPs or small insertions and deletions that corresponded with beta-lactam antibiotic resistance.

In the set of S. pneumoniae isolates from Thailand, the team detected 858 such variants, while data from the US isolates pointed to 1,721 resistance-related SNPs.

Meanwhile, a comparison of variants identified in each of the populations highlighted 301 SNPs apparently associated with beta-lactam resistance in both the Thai and American S. pneumoniae isolates, including 298 SNPs falling in predicted protein-coding sequences.

Among them were variants falling in genes encoding components of a pathway involved in making peptidoglycan material found in bacterial cell walls. Such apparent associations fit with the activity of beta-lactam antibiotics, which typically interfere with the bacterial cell wall production process.

Though more potential resistance contributors turned up in isolates from the US, the study's authors cautioned that that set likely contains false-positive "hitchhiker" variants, since the American isolates tended to have longer blocks of linked SNPs than the Thai isolates tested.

They also noted that there may have been differences in the stringency with which variant associations were called due to separate population stratification analyses in isolates from each site.

Resistance-related SNPs turned up not only in S. pneumoniae isolates resembling those targeted by existing vaccines, they noted, but also in those expected to slip past current vaccine targets.

"For the first time, we are able to see, at large scale, causative variants that allow bacteria such as Streptococcus pneumoniae to resist our efforts to treat and control it," first author Claire Chewapreecha, with the Sanger Institute, said in a statement. "We can begin to see how this might help us to develop more effective treatment strategies in the near future."