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Clostridium Difficile Speciation Occurring in Healthcare Settings, Genomic Study Suggests

NEW YORK – The diarrhea-causing enteropathogen Clostridium difficile may be undergoing active speciation in healthcare settings, according to a genomic analysis by an international team led by investigators at the Wellcome Sanger Institute.

"[W]e report the formation of an emerging C. difficile species, selected for metabolizing simple dietary sugars and producing high levels of resistant spores, that is adapted for healthcare-mediated transmission," co-senior and co-corresponding author Trevor Lawley, a host-microbiota interaction researcher at Sanger, and his co-authors wrote in their Nature Genetics study.

Using whole-genome sequencing, Lawley and his colleagues profiled more than 900 C. difficile isolates from human, animal, or environmental sources, comparing the sequences to one another and to 13 high-quality reference genomes developed from the set. With these data, they were able to track C. difficile genomic features found in various locations and hosts, uncovering signs of positive selection on core sporulation and metabolic pathway genes that seemed to contribute to speciation, further adapting the bugs to moving between individuals in healthcare setting.

"Functional validation shows that the new C. difficile produces spores that are more resistant and have increased sporulation and host colonization capacity when glucose or fructose is available for metabolism," the authors reported.

It is well known from prior research that C. difficile can form durable, long-lasting, metabolically dormant spores that help the pathogen survive and thrive in healthcare settings, the team noted. In particular, the enteropathogen is known for causing diarrhea in patients treated with antibiotics.

For their new analysis of C. difficile genome evolution, the researchers did Illumina paired-end genome sequencing on 761 C. difficile isolates from humans, 116 isolates from non-human animals, and C. difficile representatives from 29 environmental sites. For 13 isolates sequenced to high coverage, they incorporated additional Roche 454 sequence reads and optical mapping data.

Though nearly three-dozen countries were represented in the analysis, more than half of the isolates, or 465, came from the UK, while the remaining isolates spanned 32 other countries.

When they grouped the isolates based on sequences across more than 1,300 single copy core genes and clues from a handful of related outgroup species, the researchers identified four main phylogenetic lineages, PG1, PG2, PG3, and PG4, each housing isolates from a range of locations and hosts.

The PG4 lineage appeared to be particularly diverse and morphologically distinct, they noted, and could be traced back to a lineage going back an estimated 385,000 years, while the other lineages split off more recently, and appeared better adapted to producing patient infections.

"Together, these observations suggest that PG4 emerged prior to the other [phylogenetic groups] and that the PG1, PG2, and PG3 population structure started to expand just prior to the implementation of the modern healthcare system," the authors explained.

Likewise, the team's analyses of average nucleotide identity, recombination events, pseudogene profiles, substitution rates, and other features hinted that the PG1, PG2, and PG3 isolates are part of a species that is distinct from PG4. Microbes in the first three lineages showed positive selection across spore coat, spore assembly, carbohydrate metabolism, amino acid metabolism, and other genes — selection events not detected in PG4 isolates, which showed signs of positive selection across other, sulfur-related genes.

With follow-up spore formation experiments, the investigators found further evidence that the clade containing PG1, PG2, and PG3 isolates has evolved to survive on simple dietary sugars, while becoming more adept at colonizing their hosts and producing infectious spores.

"[W]e show that these recent genomic adaptations have occurred in established, distinct evolutionary lineages, each with core genomes expressing unique, pre-existing transmission properties," the authors wrote, noting that new species formation appeared to occur in microbes "primed for human transmission in the modern healthcare system."