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Genomic Study Details Emergence and Spread of Epidemic C. difficile Lineages Behind Hospital Outbreaks

NEW YORK (GenomeWeb News) – Not one but two epidemic Clostridium difficile lineages that emerged in North America are behind hospital outbreaks at centers around the world since 2001, according to a new Nature Genetics study by a Wellcome Trust Sanger Institute-led team.

"The key common factor was that antibiotic resistance emerged in both of [the lineages]," co-senior author Trevor Lawley, a bacterial pathogenesis researcher at the Sanger Institute, told GenomeWeb Daily News, explaining that this resistance made the bugs impervious to a class of very widely used antibiotics.

Based on whole-genome sequence and phylogenetic analyses on hundreds of C. difficile isolates collected around the world since the mid-1980s, Lawley and his colleagues determined that C. difficile's transformation into a dangerous and widespread healthcare-associated pathogen can be traced back to the early to mid-1990s, when two independent groups of C. difficile bacteria with the 027/BI/NAP1 genotype developed resistance to fluoroquinolone antibiotics.

"This was a bug that wasn't a major problem before," Lawley noted. "And all of a sudden it was killing people in large numbers."

Once rare, epidemic C. difficile with the 027/BI/NAP1 genotype has turned up with increasing frequency over the past decade or more.

Though some individuals colonized with C. difficile carry the pathogen for long periods of time without showing symptoms, the bug can also cause severe diarrhea, a large intestine infection called pseudomembranous colitis, and even death, authors of the new study explained.

Individuals in healthcare centers tend to be particularly prone to infection by the pathogen, which produces tenacious and long-lasting spores. So, too, are those taking antibiotics, they explained, and C. difficile 027/BI/NAP1 has become one of the top culprits in infectious diarrhea cases occurring after antibiotic treatment.

"When you take antibiotics you damage your microbiota," Lawley explained. "And [C. difficile] exploits that to colonize and cause disease."

But despite the attention and scrutiny that individual outbreaks have garnered, factors contributing to the pathogen's rise and dispersal are poorly understood.

"Although [fluoroquinolone-resistant] C. difficile 027/BI/NAP1 is widespread in healthcare facilities worldwide," the researchers wrote, "the underlying reasons for its rapid emergence and the subsequent patterns of global spread remain unknown."

To begin understanding that process, the team used paired-end Illumina HiSeq 2000 whole-genome sequencing to tackle a set of 151 C. difficile isolates that had been collected in 19 countries since 1985, mainly within hospitals.

"We pre-date the emergence of this particular [epidemic] strain, so that gives us a foundation to compare to," Lawley noted.

From the nearly 3,700 SNPs identified in the C. difficile genome sequences, researchers narrowed in on 536 phylogenetically informative variants by excluding clusters of SNPs suspected of stemming from homologous recombination events. This smaller set of SNPs was subsequently used for their phylogenetic analyses of the global strains as well as an analysis of 188 isolates from the UK, which included 43 of the isolates from the global set.

The team's analyses of the global set uncovered two epidemic C. difficile 027/BI/NAP1 lineages.

Bugs from each lineage contain the same mutation in a gene called gyrA, which codes for subunit A of a DNA gyrase enzyme, rendering them resistant to fluoroquinolone. But phylogenetic patterns indicate that the two lineages — dubbed FQR1 and FQR2 —acquired this mutation independently, roughly a year apart.

FQR1 apparently became resistant to fluoroquinolone around 1993, the team reported, subsequently contributing to outbreaks in Pittsburgh and other parts of the American northeast between 2001 and 2007, as well as infections in South Korea and Switzerland since 2007.

The other epidemic lineage, FQR2, acquired fluoroquinolone resistance roughly a year later, but traveled even further — turning up first in Montreal in 2003 and eventually spreading to healthcare centers in the UK, continental Europe, and Australia.

A spike in fluoroquinolone use in the early 2000s may have contributed to the earliest outbreaks, Lawley explained, creating conditions that favored forms of C. difficile 027/BI/NAP1 bacteria that could withstand the drug.

"Fluoroquinolone use around that time got really high, especially around 2000 and 2001," he said. "And that seems to have been the environmental driving factor for a lot of this."

Because C. difficile hadn't historically been considered a particularly dangerous hospital microbe, Lawley noted, there may have been delays between the onset of the first healthcare-associated C. difficile outbreaks and the time that each was recognized as such.

When researchers looked more closely at transmission patterns specifically within the UK, meanwhile, they saw signs that the FQR2 C. difficile 027/BI/NAP1 lineage was introduced to the UK on multiple occasions, spreading within the area and beyond.

"This collection confirms that FQR2 C. difficile 027/BI/NAP1 probably reached the UK on at least four separate occasions," they wrote, "arriving independently in Exeter, Ayrshire, and Birmingham from North America and arriving in Maidstone from continental Europe."

Together with other C. difficile 027/BI/NAP1 genome features, such patterns point to a fairly new pathogen that has spread quickly and extensively, now turning up in healthcare centers throughout the developed world.

In addition to serving as a resource during future healthcare outbreaks of C. difficile 027/BI/NAP, researchers are optimistic that the genome data they've generated for epidemic C. difficile from both lineages will help in getting a firmer handle on the bug's biology.

"Because we had two lineages that became epidemic, this actually helped us to narrow down the genetic changes that could be linked to this," Lawley explained. "So we assume that the genetic changes that happened in both lineages were important for the emergence."

Along with the fluoroquinolone resistance-related mutation in the gyrA gene, for instance, the team found that many of the isolates in the FQR1 and FQR2 lineages also contain a mobile element called Tn6192.

That element, apparently nabbed from some sort of gut microbe, is suspected of containing genes contributing to the epidemic C. difficile pathogenesis and transmission profiles, Lawely explained — a possibility that researchers are currently exploring through targeted mutation experiments.

"We can test those [mutants] in the lab and also in mouse infections to see if the mutants are less virulent or less transmissible compared to the parental strain," he said.

The team is also in the process of characterizing additional C. difficile 027/BI/NAP isolates from the healthcare setting, along with C. difficile isolates representing genotypes that are typically found outside of hospitals.

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