Scholarly Work - Center for Biofilm Engineering
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Item Search for a Shared Genetic or Biochemical Basis for Biofilm Tolerance to Antibiotics across Bacterial Species(American Society for Microbiology, 2022-04) Stewart, Philip S.; Williamson, Kerry S.; Boegli, Laura; Hamerly, Timothy; White, Ben; Scott, Liam; Hu, Xiao; Mumey, Brendan M.; Franklin, Michael J.; Bothner, Brian; Vital-Lopez, Francisco G.; Wallqvist, Anders; James, Garth A.Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for 3 days and then challenged with respective antibiotics (ciprofloxacin, daptomycin, and tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells, and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of individual microorganisms in biofilms and contribute to antibiotic tolerance.Item Analysis of Clostridium difficile biofilms: imaging and antimicrobial treatment(2018-01) James, Garth A.; Chesnel, L.; Boegli, Laura; Pulcini, Elinor D.; Fisher, Steve T.; Stewart, Philip S.BACKGROUND: Clostridium difficile, a spore-forming Gram-positive anaerobic bacillus, is the most common causative agent of healthcare-associated diarrhoea. Formation of biofilms may protect C. difficile against antibiotics, potentially leading to treatment failure. Furthermore, bacterial spores or vegetative cells may linger in biofilms in the gut causing C. difficile infection recurrence. OBJECTIVES: In this study, we evaluated and compared the efficacy of four antibiotics (fidaxomicin, surotomycin, vancomycin and metronidazole) in penetrating C. difficile biofilms and killing vegetative cells. METHODS: C. difficile biofilms grown initially for 48 or 72 h using the colony biofilm model were then treated with antibiotics at a concentration of 25 × MIC for 24 h. Vegetative cells and spores were enumerated. The effect of treatment on biofilm structure was studied by scanning electron microscopy (SEM). The ability of fidaxomicin and surotomycin to penetrate biofilms was studied using fluorescently tagged antibiotics. RESULTS: Both surotomycin and fidaxomicin were significantly more effective than vancomycin or metronidazole (P < 0.001) at killing vegetative cells in established biofilms. Fidaxomicin was more effective than metronidazole at reducing viable spore counts in biofilms (P < 0.05). Fluorescently labelled surotomycin and fidaxomicin penetrated C. difficile biofilms in < 1 h. After 24 h of treatment, SEM demonstrated that both fidaxomicin and surotomycin disrupted the biofilm structure, while metronidazole had no observable effect. CONCLUSIONS: Fidaxomicin is effective in disrupting C. difficile biofilms, killing vegetative cells and decreasing spore counts.Item Effects of ultrasonic treatment on the efficacy of gentamicin against established pseudomonas aeruginosa biofilms(1996-05) Huang, Ching-Tsan; James, Garth A.; Pitt, William G.; Stewart, Philip S.The effect of simultaneous ultrasonic treatment on the efficacy of gentamicin against planktonic and established biofilm cells of Pseudomonas aeruginosa was investigated. Planktonic cells were treated with 6 or 12 μg ml−1 of gentamicin for 4 h with ultrasonic treatment at three levels of power density (0.2, 2 and 15 mW cm−2). Biofilm cells grown on stainless steel slides in a continuous flow reactor were treated with 30 μg ml−1 of gentamicin and ultrasound. Ultrasound itself at these power levels did not cause cell killing or lysis in planktonic and biofilm cultures. Concentrations of 6 and 12 mg ml−1 gentamicin led to 2.65- and 2.75-log reductions of the surviving fraction in planktonic cultures in the absence of ultrasound. The addition of ultrasound did not show further reduction compared with those without ultrasonication. Gentamicin (30 μg ml−1) caused variable killing in biofilms which ranged from 0.83- to 2.86-log reductions of the surviving fraction without ultrasonication. Gentamicin efficacy measured by the surviving fraction was improved by 0.28-, 1.12- and 0.58-log when coupled with 0.2, 2 and 15 mW cm−2 ultrasonic treatments, respectively. Experimental results indicated that ultrasound modestly improved the efficacy of gentamicin against established P. aeruginosa biofilms.Item Letter to the Editor: Highlights from the Montana wound biofilm retreat(2009-07) Ammons, Mary Cloud B.; James, Garth A.; Stewart, Philip S.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 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.Item 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 wounds—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.Item Propionibacterium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy(2017-04) Capoor, Manu N.; Ruzicka, Filip; Schmitz, Jonathan E.; James, Garth A.; Machackova, Tana; Jancalek, Radim; Smrcka, Martin; Lipina, Radim; Ahmed, Fahad S.; Alamin, Todd F.; Anand, Neel; Baird, John C.; Bhatia, Nitin; Demir-Deviren, Sibel; Eastlack, Robert K.; Fisher, Steve T.; Garfin, Steven R.; Gogia, Jaspaul S.; Gokaslan, Ziya L.; Kuo, Calvin C.; Lee, Yu-Po; Mavrommatis, Konstantinos; Michu, Elleni; Noskova, Hana; Raz, Assaf; Sana, Jiri; Shamie, A. Nick; Stewart, Philip S.; Stonemetz, Jerry L.; Wang, Jeffrey C.; Witham, Timothy F.; Coscia, Michael F.; Birkenmaier, Christof; Fischetti, Vincent A.; Slaby, OndrejBackground In previous studies, Propionibacterium acnes was cultured from intervertebral disc tissue of ~25% of patients undergoing microdiscectomy, suggesting a possible link between chronic bacterial infection and disc degeneration. However, given the prominence of P. acnes as a skin commensal, such analyses often struggled to exclude the alternate possibility that these organisms represent perioperative microbiologic contamination. This investigation seeks to validate P. acnes prevalence in resected disc cultures, while providing microscopic evidence of P. acnes biofilm in the intervertebral discs. Methods Specimens from 368 patients undergoing microdiscectomy for disc herniation were divided into several fragments, one being homogenized, subjected to quantitative anaerobic culture, and assessed for bacterial growth, and a second fragment frozen for additional analyses. Colonies were identified by MALDI-TOF mass spectrometry and P. acnes phylotyping was conducted by multiplex PCR. For a sub-set of specimens, bacteria localization within the disc was assessed by microscopy using confocal laser scanning and FISH. Results Bacteria were cultured from 162 discs (44%), including 119 cases (32.3%) with P. acnes. In 89 cases, P. acnes was cultured exclusively; in 30 cases, it was isolated in combination with other bacteria (primarily coagulase-negative Staphylococcus spp.) Among positive specimens, the median P. acnes bacterial burden was 350 CFU/g (12 - ~20,000 CFU/g). Thirty-eight P. acnes isolates were subjected to molecular sub-typing, identifying 4 of 6 defined phylogroups: IA1, IB, IC, and II. Eight culture-positive specimens were evaluated by fluorescence microscopy and revealed P. acnes in situ. Notably, these bacteria demonstrated a biofilm distribution within the disc matrix. P. acnes bacteria were more prevalent in males than females (39% vs. 23%, p = 0.0013). Conclusions This study confirms that P. acnes is prevalent in herniated disc tissue. Moreover, it provides the first visual evidence of P. acnes biofilms within such specimens, consistent with infection rather than microbiologic contamination.Item Bacterial biofilm in acute lesions of hidradenitis suppurativa(2017-01) Okoye, Ginette A.; Vlassova, Natalia; Olowoyeye, Omolara; Agostinho, Alessandra; James, Garth A.; Stewart, Philip S.; Leung, Anthony; Lazarus, Gerald S.
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