Browsing by Author "Usui, Marcia L."

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Biofilms and inflammation in chronic wounds
(2013-09) Zhao, Ge; Usui, Marcia L.; Lippman, S. I.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
SIGNIFICANCE: The incidence, cost, morbidity, and mortality associated with non-healing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair.RECENT ADVANCES: Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity.CRITICAL ISSUES: When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host.FUTURE DIRECTIONS: Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy.
Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: A model for the study of chronic wounds
(2010-08) Zhao, Ge; Hochwalt, Phillip C.; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
Chronic wounds are a major clinical problem that lead to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models.The goal of this study was to create a reproducible chronic wound model in diabetic mice by the application of bacterial biofilm. Six-millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms 2 days postwounding, and covered with semiocclusive dressings for 2 weeks. Most of the control wounds were epithelialized by 28 days postwounding. In contrast, none of biofilm-challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged woundsâ€”all indicators of an inflammatory nonhealing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality, and demonstrated delayed wound healing following a biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing.
Development of a chronic wound in a diabetic (db/db) mouse by infection with biofilm
(2008) Zhao, Ge; Hochwalt, Phillip C.; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
Chronic wounds such as diabetic ulcers, pressure sores, and venous stasis ulcers are a major source of morbidity and mortality. Since chronic ulcers are not homogeneous, systematic study of ulcer therapies is difficult. Our goal is to create a standard chronic wound model. It has been shown that specialized microbial colonies known as biofilm are present in chronic human wounds. The microorganisms present in biofilm are protected from the host defenses, topical antiseptics, and systemic antibiotics. For this reason, biofilm infections persist and may contribute to poor wound healing. We hypothesize that application of biofilm to an already characterized diabetic mouse wound model may create a reproducible chronic wound. Bacterial biofilm was developed by incubating planktonic Pseudomonas aeruginosa (PAO-1) and transferring to polycarbonate membrane filters placed on LB agar plates. The biofilm (~10^8 CFU) was transferred to 6mm wounds created on the dorsal skin of diabetic mice and the wounds were covered with Tegaderm® dressing. The biofilm was transferred to the wounds at several different time points following wounding. The mice died if the biofilm was transferred soon after wounding; however, mice that were allowed to recover from the surgery before biofilm inoculation developed a purulent wound that persisted for weeks. In addition, the timing of Tegaderm® application proved to be a critical variable for purulence and quality of the wound. Ultimately, the development of a reproducible chronic wound in a diabetic mouse will allow in vivo testing of potential wound healing therapies.
Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.
(2016-04) James, Garth A.; Zhao, Alice Ge; Usui, Marcia L.; Underwood, Robert A.; Nguyen, Hung; Beyenal, Haluk; Pulcini, Elinor D.; Hunt, Alessandra Agostinho; Bernstein, Hans C.; Fleckman, Philip; Olerud, John E.; Williamson, Kerry S.; Franklin, Michael J.; Stewart, Philip S.
Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.
Resident bacterial flora in the skin of C57BL/6 mice housed under SPF conditions
(2010-09) Tavakkol, Zarry; Samuelson, Derrick; Pulcini, Elinor D.; Underwood, Robert A.; Usui, Marcia L.; Costerton, J. William; James, Garth A.; Olerud, John E.; Fleckman, Philip
Research in cutaneous biology frequently involves models that use mice housed in SPF conditions. Little information is available concerning the species of bacteria that normally inhabit the skin of these mice. The aim of this study was to characterize the bacterial skin flora of mice housed under SPF conditions. Skin biopsies from C57BL/6 mice under normal and surgically prepped conditions were both cultured and analyzed by using DNA extraction and sequencing. The species isolated most commonly from culture were staphylococci. Coagulase-negative staphylococci were isolated more frequently than was Staphylococcus aureus. Molecular sequencing yielded several additional organisms not found by culture. Overall, culturing of isolates yielded 14 species of bacteria, and molecular sequencing identified another 6 species. Investigators conducting cutaneous research in mouse models should aware of the cutaneous bacterial flora present on these mice.
Time course study of delayed wound healing in a biofilm-challenged diabetic mouse model
(2012-05) Zhao, Ge; Usui, Marcia L.; Underwood, Robert A.; Singh, Pradeep K.; James, Garth A.; Stewart, Philip S.; Fleckman, Philip; Olerud, John E.
Bacterial biofilm has been shown to play a role in delaying wound healing of chronic wounds, a major medical problem that results in significant health care burden. A reproducible animal model could be very valuable for studying the mechanism and management of chronic wounds. Our previous work showed that Pseudomonas aeruginosa (PAO1) biofilm challenge on wounds in diabetic (db/db) mice significantly delayed wound healing. In this wound time course study, we further characterize the bacterial burden, delayed wound healing, and certain aspects of the host inflammatory response in the PAO1 biofilm-challenged db/db mouse model. PAO1 biofilms were transferred onto 2-day-old wounds created on the dorsal surface of db/db mice. Control wounds without biofilm challenge healed by 4 weeks, consistent with previous studies; none of the biofilm-challenged wounds healed by 4 weeks. Of the biofilm-challenged wounds, 64% healed by 6 weeks, and all of the biofilmchallenged wounds healed by 8 weeks. During the wound-healing process, P. aeruginosa was gradually cleared from the wounds while the presence of Staphylococcus aureus (part of the normal mouse skin flora) increased. Scabs from all unhealed wounds contained 107 P. aeruginosa, which was 100-fold higher than the counts isolated from wound beds (i.e., 99% of the P. aeruginosa was in the scab). Histology and genetic analysis showed proliferative epidermis, deficient vascularization, and increased inflammatory cytokines. Hypoxia inducible factor expression increased threefold in 4-week wounds. In summary, our study shows that biofilm-challenged wounds typically heal in approximately 6 weeks, at least 2 weeks longer than nonbiofilm-challenged normal wounds. These data suggest that this delayed wound healing model enables the in vivo study of bacterial biofilm responses to host defenses and the effects of biofilms on host wound healing pathways. It may also be used to test antibiofilm strategies for treating chronic wounds.