Tuesday, 06 September 2016 14:59

Diverse fungi inhabit chronic wounds and inhibit healing

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Published in mBiosphere

Gruesome, ghastly, grisly. These are the words that popped into my head when I googled images of diabetic foot ulcers—one of the most common chronic wounds creating a silent and costly epidemic in healthcare. 

Perhaps even more shocking is the mortality rate connected to these open wounds, which are often caused by pressure or injury and exacerbated by the vascular and nerve degeneration that diabetics experience in their extremities. “The 5-year mortality rate associated with these ulcers is around 45-55 percent. That’s higher than the rate for Hodgkin’s lymphoma, breast cancer, or prostate cancer—which really puts it into context,” says Elizabeth Grice, a microbiologist at the University of Pennsylvania.  

Up to one-quarter of the 29 million Americans with diabetes will experience a DFU in their lifetime, says Grice. A sign of serious illness, these wounds are difficult to manage, severely limit mobility, and can lead to complications such as bone infections, amputations, and even sepsis and death.

Grice and postdoctoral researcher Lindsay Kalan were particularly intrigued by the lack of information on what fungal species might be present in the microbiome of these ulcers—and whether they impede healing by forming biofilms or in other ways. This week in mBio their team, along with collaborators at University of Iowa, reveals the first deep look at the mycobiome of a chronic wound. They found fungus is among us in chronic wounds—fungal communities were both prevalent and predictive of delayed healing.

“It’s thought that microbes impair healing in chronic wounds because they form a biofilm,” says Grice. Kalan continues, “Physically, biofilms produce an extracellular matrix, or slime, that prevents the penetration of antimicrobial drugs. Within the biofilm, microbial cells can have lower rates of metabolism that make them more tolerant to antimicrobials as well.”

Grice and Kalan, along with Sue Gardner in Iowa, decided to follow 100 diabetic foot ulcers (DFUs) for six months, or until the ulcer healed or resulted in an amputation. Gardner, an associate professor of nursing, took samples of the deep fluid in patients’ wounds every two weeks during treatment. Each patient received the same standard care—cleansing and debriding of the wound, followed by offloading pressure from the foot with a cast or boot.  Gardner then sent the samples to Grice and Kalan for genetic sequencing.

From those samples, Kalan then sequenced the nuclear ribosomal internal transcribed spacer 1 (ITS1), a ribosomal sequence that serves as the genetic barcode for different fungal species. Previous studies based on culturing fungal species from wounds in the lab had estimated that 25-30 percent of wounds harbored fungus. But Kalan found that 80 percent of the DFUs contained at least one fungus.

There are severe limitations in the fungal sequence databases—almost a third of the fungal sequences could only be classified at the Kingdom level. Even so, the study revealed a surprising diversity of at least 284 fungal species living deep inside DFUs. The team found that the most frequently found species were Cladosporium herbarum (in 41 percent of samples) and Candida albicans (in 22 percent).

The mix of fungi found included human pathogens like C. albicans and Aspergillus, but lacked the most common fungus found living on human skin, Malassezia. Kalan says finding C. herbarum was most dominant was a surprise: “This is one of the most ubiquitous fungi on the planet, found everywhere and its spores can cause common mold allergies.  But generally, it’s not thought to cause skin or wound infections.”

When the team compared the fungal communities found in each wound with the patients’ outcomes, it did not appear that single fungal pathogens were driving bad outcomes, but rather patients with non-healing or amputated wounds had mixed communities of fungi.

Taking an even closer look, Kalan chose two patients who had pathogenic fungi present for two consecutive visits—one whose DFU took greater than six weeks to heal and one whose DFU resulted in an amputation—and cultured their microbes in the lab. The delayed healing patient harbored both C. albicans fungus and a bacterium, Citrobacter freundii.  The amputation patient’s wound held the fungus Trichosporon asahii and Staphylococcus simulans bacteria.  When Kalan co-cultured these bacterial-fungal pairs in the lab dish, she found that they formed intertwined biofilms within 1-2 days.

“These two interactions haven’t been described before,” says Kalan. “The fungal and bacterial cells are attached and adhered together, not just simply hanging out in the same area. If you zoom in, you can see the fungal hyphae coated with bacterial cells.”

Grice says the work shows that “fungi are an important component of the human microbiome and can’t be ignored when looking at the pathogenesis of chronic wounds.”  Although the molecular sequencing tests that her team used aren’t available yet in the doctor’s office, they might be someday. This work demonstrates that categorizing the bacterial and fungal players involved in a chronic wound will be a key first step for doctors to plan their best strategy for healing wounds quickly and effectively.

Last modified on Tuesday, 08 November 2016 16:15
Kendall Powell

Freelance science writer and editor, Kendall Powell covers the realm of biology, from molecules to maternity. She has written news stories, features and scientist profiles for a variety of publications including the Washington Post, Los Angeles Times, Nature, PLoS Biology, Journal of Cell Biology, Science Careers and the HHMI Bulletin. She is a contributor to The Science Writers’ Handbook: Everything You Need to Know to Pitch, Publish, and Prosper in the Digital Age (2013 Da Capo).