Publications by Colleges and Departments (MSU - Bozeman)
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Item Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro(2009-09) Kirker, Kelly R.; Secor, Patrick R.; James, Garth A.; Fleckman, Philip; Olerud, John E.; Stewart, Philip S.Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies demonstrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insert-supported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendritelike extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (p 0.0004). At 24 hours, biofilm-, BCM-, and PCM-exposed HK all exhibited reduced scratch closure (p 0.0001). The results demonstrated that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus. Keratinocytes are the major cell type of the epidermis, which serves as the primary barrier between the external environment and the internal tissues. In this capacity, the epidermis also functions as a barricade to microorganisms, toxins, and various antigens. When the barrier is breached due to wounding, basal keratinocytes from the wound edges or dermal appendages migrate over the open wound to reestablish the barricade in a process called reepithelialization. Chronic wounds, such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers, are characterized by prolonged inflammation, an altered wound matrix, and the failure to reepithelialize. Chronic wounds are also characterized as supporting a diverse microbial flora. A literature review by Bowler examined culture data from 62 published studies dating between 1969 and 1997.1 The most predominant wound isolate in both chronic and acute wounds was Staphylococcus aureus (reported in 63% of the studies), followed by coliforms (45%), Bacteroides spp. (39%), Peptostreptococcus spp. (36%), Pseudomonas aeruginosa (29%), Enterococcus spp. (26%), and Streptococcus pyogenes (13%).1 Using molecular techniques, Dowd et al.2 also demonstrated vast bacterial diversity within chronic wounds. The most prevalent species included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. It has been speculated that bacteria colonizing chronic wounds exist as a biofilm.3–7 Biofilms represent bacterial communities surrounded by extracellular polysaccharide matrix. Such communities are often polymicrobial and resistant to antimicrobials. Chronic wounds are an ideal environment for bacterial infection and biofilm formation. The wound remains open for a prolonged period of time, increasing the odds of bacterial infection. The wound bed provides a surface for growth, and poor blood flow and hypoxia discourage native defenses.8 Studies have shown that wounds inoculated with bacteria form biofilms.6,9 Furthermore, in a recent study by James et al.,10 60% of chronic wound specimens were characterized as containing biofilm compared with 6% of acute wound specimens. Despite the prevalence of biofilms in wounds, there are few data illustrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for directly studying the effect of biofilms on the cell types involved in wound healing. Specifically, it examines the effect of S. aureus biofilms on keratinocyte morphology, viability, and scratch closure. METHOD ANDMATERIALS Cell culture Normal human keratinocytes (HK) were isolated from newborn foreskin using methods previously described11 and in accordance with the University of Washington 690 WoundItem Biofilms in chronic wounds(2008-01) James, Garth A.; Swogger, E.; Wolcott, Randall D.; Pulcini, Elinor D.; Secor, Patrick R.; Sestrich, Jennifer; Costerton, J. William; Stewart, Philip S.Chronic wounds including diabetic foot ulcers, pressure ulcers, and venous leg ulcers are a worldwide health problem. It has been speculated that bacteria colonizing chronic wounds exist as highly persistent biofilm communities. This research examined chronic and acute wounds for biofilms and characterized microorganisms inhabiting these wounds. Chronic wound specimens were obtained from 77 subjects and acute wound specimens were obtained from 16 subjects. Culture data were collected using standard clinical techniques. Light and scanning electron microscopy techniques were used to analyze 50 of the chronic wound specimens and the 16 acute wound specimens. Molecular analyses were performed on the remaining 27 chronic wound specimens using denaturing gradient gel electrophoresis and sequence analysis. Of the 50 chronic wound specimens evaluated by microscopy, 30 were characterized as containing biofilm (60%), whereas only one of the 16 acute wound specimens was characterized as containing biofilm (6%). This was a statistically significant difference (p<0.001). Molecular analyses of chronic wound specimens revealed diverse polymicrobial communities and the presence of bacteria, including strictly anaerobic bacteria, not revealed by culture. Bacterial biofilm prevalence in specimens from chronic wounds relative to acute wounds observed in this study provides evidence that biofilms may be abundant in chronic wounds.Item Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing(2008-03) Dowd, Scot E.; Sun, Yan; Secor, Patrick R.; Rhoads, Daniel D.; Wolcott, Benjamin M.; James, Garth A.; Wolcott, Randall D.BACKGROUND:Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate 16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D), venous leg ulcers (V), and pressure ulcers (P). RESULTS:There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods.CONCLUSIONS: If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in order to improve the outcomes of patients. This survey will also foster the pioneering and development of new molecular diagnostic tools, which can be used to identify the community