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Sanger Institute Sequences 240 Pneumococcal Isolates to Track Evolution of Drug Resistance

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By Monica Heger

This story was originally published Feb. 3.

Sequencing bacterial isolates could help predict epidemics, identify new drug targets, and monitor drug resistance, according to a study by the Wellcome Trust Sanger Institute.

Sanger researchers recently sequenced the whole genomes of 240 isolates of the bacteria Streptococcus penumoniae, the pathogen responsible for pneumonia, meningitis, and other diseases, in order to piece together how the pathogen has evolved, mutated, and built up resistance to drugs.

The study, published in January in Science, shows how next-generation sequencing can be used to identify variants that allow pathogenic bacteria to evade drugs, and could enable the development of new therapies.

The sequencing allowed the team to "see in more detail how the [pathogen] spread from country to country and how the resistance was acquired," Stephen Bentley, a senior staff scientist at the Sanger Institute who led the research, told In Sequence.

Bacteria is unique in that it not only frequently acquires point mutations, but it also exchanges genetic information with other strains, including elements responsible for pathogenicity, said Elena Jouravleva, global product manager at Beckman Coulter Genomics, who was not involved with the study. Therefore these types of analyses are important for monitoring pathogens.

The Sanger researchers sequenced on the Illumina Genome Analyzer multiplexed genomic DNA libraries of 240 isolates from a lineage that has developed resistance to penicillin and other antibiotics. The strains had been isolated between 1984 and 2008.

They created libraries with a 200 base pair insert size and used a paired-end sequencing strategy with 54 base pair reads, sequencing 96 isolates, 12 per lane, in one run on the GA. All samples were sequenced to an average of 9-fold coverage, with 73.4-fold coverage across all samples.

When mapping to the reference, they identified 57,736 SNPs. By comparing SNPs that were formed from recombination events to those that were not, they were able to estimate that mutations outside of recombination occur at a rate of approximately 1.57 in 10 million per site per year.

Analyzing the mutations that accrued outside of recombination also allowed them to piece together a more accurate phylogenetic tree, showing that the lineage emerged in the 1970s, after the introduction of penicillin and other antibiotics to which the strain is resistant. Including the recombinant mutations incorrectly dated the lineage as emerging in the 1930s.

Aside from base substitutions, the researchers detected 1,032 small insertions and deletions, 61 percent of which are located in the 13 percent of the genome that is non-coding.

Strikingly, they found that 74 percent of the reference genome length had undergone recombination in at least one isolate, with hotspots occurring in areas that are targeted by human antibodies. "It seems likely that these loci are under diversification selection driven by the human immune system," the authors wrote.

The researchers said that understanding the rate of mutation, the types of mutations that accumulate, and recombination patterns will lead to better surveillance tools to monitor disease spread and predict epidemics, and also better patient diagnosis and drugs.

Bentley said the most immediate application would be in surveillance. For example, a database of sequenced pathogen genomes would help researchers predict epidemics by identifying clones that are spreading rapidly through the population.

"Pathogen evolution is being driven by its interaction with clinical practices. If we can continue to monitor that evolution using whole-genome sequencing we can see the impact that we're having, and adjust, and maybe come up with new [treatment] approaches," Bentley said.

Jouravleva agreed and said that Beckman Coulter has seen an increase in requests from its customers to use whole-genome sequencing to evaluate drug resistance in bacteria. Being able to monitor drug resistance could allow clinicians to make decisions about when to discontinue the use of an antibiotic or treatment, she said. Eventually, pathogen sequencing could be done "at the bedside of the patient to make decisions about treatment," she added.

The Sanger team's next steps are to do similar studies on other bacterial pathogen species, and also more studies within the pneumococcal species. He said they want to use sequencing to understand disease transmission, as well as why particular clones are more successful at spreading and why some have remained prevalent in the population despite not having acquired antibiotic resistance.


Have topics you'd like to see covered in In Sequence? Contact the editor at mheger [at] genomeweb [.] com.

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