LONDON – The largest-ever genomics study of Clostridium difficile has found an emerging new species of the bacterium is selected to thrive on a Western sugar-rich diet and to produce high levels of resistant spores, adapting it to maximize transmission in hospitals and other health care facilities.

The researchers found changes in genes that enable the emerging species, C. difficile clade A, to metabolize sucrose and fructose, and showed the bacterium better colonized mice when their diet was enriched with sugar.

C. difficile clade A also has evolved differences in sporulation genes, leading to the formation of spores that are resistant to a common hospital disinfectant, and allowing it to spread more easily in health care settings.

Over the past four decades, C. difficile has emerged as the leading cause of antibiotic-associated diarrhea worldwide. The genomic analysis shows its recent and rapid emergence as a significant health care pathogen is not only a result of acquiring antibiotic resistance via horizontal gene transfer, but that human lifestyles are driving C. difficile to form a new species, enabling it to spread more easily.

Nitin Kumar, of the Wellcome Sanger Institute in Cambridge, U.K., an author of the paper published in the Aug. 12, 2019, online edition of Nature Genetics, said C. difficile clade A "was primed to take advantage of modern health care practices and human diets before hospitals even existed."

Screens show that C. difficile clade A makes up 68.5% of samples from U.S. hospital patients; 74% of samples in Europe and 100% in China.

To define the evolutionary history and genetic changes underpinning the emergence of C. difficile as a health care-associated pathogen, the researchers brought together and sequenced the whole genomes of the biggest collection of C. difficile to date, amassing 906 strains from 33 countries. Of those, 761 were isolated from humans, 116 from animals and 29 from environmental sources.

The collection was designed to comprehensively capture C. difficile diversity, with four major phylogenetic groups represented. Each harbored strains from different geographical locations, hosts and environmental sources, indicating sympatric speciation.

Analysis of the four phylogenetic groups showed groups one, two and three were descended from phylogenetic group four, and that populations of groups one through three started to expand around 1595 AD, shortly before the emergence of the modern health care system in the eighteenth century. Phylogenetic groups one through three form the emerging species, C. difficile clade A.

"This emerging species has existed for thousands of years, but this is the first time anyone has studied C. difficile genomes in this way to identify it," Kumar said. "Our large-scale genetic analysis allowed us to discover that C. difficile is currently forming a new species, with [clade A] specialized to spread in hospital environments."

The researchers identified 172 core genes in clade A that were positively selected. Among those are genes involved in spore coat architecture, spore coat assembly and metabolizing sugars.

In total, 45 (26%) of the positively selected genes produce proteins that are directly involved in spore production in clade A. By contrast, no positively selected genes are directly involved in spore production in clade B, suggesting that functions that are important for host-to-host transmission have evolved in clade A.

When spores of both clades were exposed to hydrogen peroxide, spores from clade A were more resistant to 3% and 10% concentrations of the widely used hospital disinfectant than those from clade B. There was no difference in survival at 30% peroxide, which had an overpowering bactericidal effect.

Clade A genomes contain genes under positive selection that are involved in metabolizing sugars, including fructose, glucose, sorbitol and ribulose. Based on that, the researchers reasoned that dietary glucose could have an impact on host colonization by spores from clade A.

They showed that mice challenged with clade A spores had a higher bacterial load when fed additional glucose or fructose, but there was no difference between clade A and clade B sores in mice without sugar supplements.

The resistant spores formed by clade A can remain on surfaces and spread easily between people, with the degree of infectivity and transmission within health care settings driven by spore density. C difficile clade A exhibited increased spore production when supplemented with glucose or fructose, in vitro.

"Our study provides genome and laboratory-based evidence that human lifestyles can drive bacteria to form new species so they can spread more effectively," said Trevor Lawley, senior author from the Wellcome Sanger Institute. "Strains of C. difficile bacteria have continued to evolve in response to modern diets and health care systems," he said. "Focusing on diet and looking for new disinfectants could help in the fight against this bacterium."

Taken together, the researchers say their findings reveal the ongoing formation of a new species with biological and phenotypical properties "consistent with a transmission cycle that is primed for human transmission in the modern health care system."

The research gives a whole new understanding of bacterial evolution and reveals the importance of genomic surveillance of bacteria, said study author Brendan Wren, of the London School of Hygiene & Tropical Medicine. "Ultimately, this could help understand how other dangerous pathogens evolve by adapting to changes in human lifestyles and health care regimes," he said.

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