"Biofilms pose an intractable problem for wound healing," says Anne Han of the Johns Hopkins Bayview Medical Center in Baltimore, Md. Microbial biofilms that form at wound sites tend to resist treatment and thus prove all the more damaging to patients, she said during the Montana Biofilm Science & Technology meeting, held in July at the Center for Biofilm Engineering (CBE) in Bozeman, Mont.

However, Han and other experts point out, by recognizing the key role biofilms play in chronic wounds, researchers are generating insights that might help to mitigate this important medical problem that now costs the U.S. health care system $25 billion yearly. Chronic wounds are particularly devastating for people with diabetes, affecting about 12% of the 20 million diabetics in the U.S.

One important step is to learn more about the typical microbial constituents in wound-associated biofilms, Han says. Conventional culturing of such sites vastly underestimates the species that collect in chronic wounds, she finds, pointing to her recent results analyzing 16S ribosomal RNA (rRNA) sequences of wound-site samples from patients with diabetes. Chronic wounds are particularly rich with anaerobic bacteria, such as Bacteroidetes and Fusobacterium spp., setting them apart from the commensal bacterial associated with normal skin. Moreover, levels of the quorum-sensing molecule AI-2 run 100 to 1,000 times higher in wound samples than in normal skin, suggesting that bacteria in wounds actively communicate among themselves, she says.

Researchers are studying wound healing in diabetic mice to better understand what goes wrong among patients with this disease, according to Ge Alice Zhao at the University of Washington, Seattle. "The goal is to create a chronic wound model in animals to study the histopathology of delayed wound healing," she says. Small skin wounds in 8-week-old diabetic mice that are inoculated with P. aeruginosa biofilms take six to eight weeks to heal, whereas identical wounds in uninfected mice heal within four weeks, she finds. This time frame provides clues about when to apply antimicrobials to optimize healing.

The majority of biofilm bacteria concentrate within wound scabs, according to Zhao. Uninfected skin of biofilm-treated mice contains 103 to 104 bacteria, compared to 108 bacterial cells in scabs. Transmission electron microscopy reveals an abundance of neutrophils in scabs, with pockets of biofilms forming a matrix around them.

Wound-derived keratinocytes, a kind of skin cell, respond differently when treated with medium in which bacterial biofilms formed versus medium in which planktonic cells were grown, according to studies by Pat Secor of CBE. For instance, biofilm medium strikingly decreases keratinocyte viability and greatly increases their likelihood of undergoing apoptosis, or programmed cell death. Additionally, exposure to biofilm medium leads keratinocyte nuclei to fragment and for some cells to form dendritic extensions. Biofilm medium also upregulates genes for cytokines and chemokines associated with inflammation. Exposure to biofilms appears "more detrimental to wound healing" than is exposure to planktonic bacteria, says Garth James, Medical Projects Manager at CBE.

Debriding chronic wounds is a standard clinical procedure, one that implicitly presumes that physically removing damaged tissues carrying biofilms will increase the antibiotic susceptibility of bacteria that remain at the treated site. However, findings in CBE wound-model studies raise doubts about how well debridement works. Thus, P. aeruginosa and S. aureus biofilms were separately grown in drip flow reactors, and then mostly removed to simulate debridement of a biofilm. However, neither biofilm proved any more susceptible to gentamicin after this simulated debridement.

The drip flow reactor is also being used to evaluate commercial wound dressings, according to James. Only products impregnated with silver or organic antimicrobials such as polyhexamethylene biguanide substantially reduce bacterial growth, he says, summarizing findings that appear in the June 2010 Journal of Wound Care.

Carol Potera
Carol Potera is a freelance writer in Great Falls, Mont.