Publications by Colleges and Departments (MSU - Bozeman)
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Item Measurements of accumulation and displacement at the single cell cluster level in Pseudomonas aeruginosa biofilms(2008-09) Klayman, Benjamin J.; Klapper, Isaac; Stewart, Philip S.; Camper, Anne K.Quantitative descriptions of biofilm growth and dynamics at the individual cell level are largely missing from the literature. To fill this gap, research was done to describe growth, accumulation and displacement patterns in developing Pseudomonas aeruginosa biofilms. A parent strain of PAO1 was labelled with either a cyan or yellow fluorescent protein. These were then grown in a flow cell biofilm together so that pockets of dividing cells could be identified and their accumulation and displacement tracked. This analysis revealed a pattern of exponential accumulation for all clusters followed by a stationary accumulation phase. A background ‘carpet’ layer of cells uniformly colonizing the surface exhibited zero net accumulation of bio-volume. The individual clusters were found to have a mean accumulation rate of 0.34 h-1 with a range of 0.28–0.41 h-1. Cluster accumulation rates were negatively correlated with cluster size; larger clusters accumulated volume at a slower rate (P < 0.001). Pockets of cells on the inside of clusters initially accumulated at a comparable rate to the cluster within which they resided, but later invariably exhibited zero to slightly negative accumulation despite continued exponential (positive) accumulation of the cluster. Expanding clusters were able to displace neighbouring cells from the surface, and larger clusters displaced smaller clusters. This work provides a more detailed quantitative experimental observation of biofilm behaviour than has been described previously.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 Biofilm maturity studies indicate sharp debridement opens a time-dependent therapeutic window(2010-08) Wolcott, Randall D.; Rumbaugh, Kendra P.; James, Garth A.; Schultz, Gregory; Phillips, P.; Yang, Q.; Watters, C.; Stewart, Philip S.; Dowd, Scot E.Objective: To investigate the hypothesis that newly formed wound biofilms (or bioburdens) are more susceptible to antimicrobial treatment.Method: Four separate and distinct models were performed by four separate biofilm research laboratories to evaluate the resistance of biofilms to antimicrobial treatments over time. These included a drip-flow biofilm model along with a hydrodebridement study, a porcine skin punch biopsy ex vivo model, a mouse chronic wound model and clinical longitudinal debridement study.Results: All four models showed that, within the first 24 hours, the biofilm community was more susceptible to the selected antibiotics, and after maturing for up to 48 hours became increasingly tolerant. In each model, there was at least a 24-hour period in which the biofilms were more resistant to antibiotics. Each of the models utilised showed a significant decrease in the resistance of the biofilm/ burden to gentamicin for up to 24 hours with a confidence interval of at least 95%. The resistance increased in each of the models by 48 hours and reached original resistance levels by 72 hours.Conclusion: These data suggest the principles of biofilm-based wound care, along with the use of serial debridement to continually remove mature biofilm, followed by biofilm wound management strategies, including topical antibiotics while the bioburden is still immature and more susceptible, are valid.Conflict of interest: SED is director of Research and Testing Laboratory, a commercial laboratory that develops molecular methods for diagnosis of wounds and infections and CEO of Pathogenius Laboratories, which is a molecular pathogen diagnostic company with a focus on chronic wounds. RDW is medical director of Southwest Regional Wound Care Center and inventor of biofilm-based wound care principles.Item Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by Pseudomonas aeruginosa using a library of truncated fragments(2012-11) Nagant, C.; Pitts, Betsey; Nazmi, K.; Vandenbranden, M.; Bolscher, J. G.; Stewart, Philip S.; Dehaye, J. P.Persistent Pseudomonas aeruginosa infections are a major cause of morbidity and mortality in cystic fibrosis (CF) patients andare linked to the formation of a biofilm. The development of new biofilm inhibition strategies is thus a major challenge. LL-37 isthe only human antimicrobial peptide derived from cathelicidin. The effects on the P. aeruginosa PAO1 strain of synthetic truncatedfragments of this peptide were compared with the effects of the original peptide. Fragments of LL-37 composed of 19 residues(LL-19, LL13-31, and LL7-25) inhibited biofilm formation. The strongest antibiofilm activity was observed with the peptidesLL7-37 and LL-31, which decreased the percentage of biomass formation at a very low concentration. Some peptides werealso active on the bacteria within an established biofilm. LL7-31, LL-31, and LL7-37 increased the uptake of propidium iodide(PI) by sessile bacteria. The peptide LL7-37 decreased the height of the biofilm and partly disrupted it. The peptides active within the biofilm had an infrared spectrum compatible with an -helix. LL-37, but not the peptides LL7-31 and LL7-37, showed cellular toxicity by permeabilizing the eukaryotic plasma membrane (uptake of ethidium bromide and release of lactate dehydrogenase [LDH]). None of the tested peptides affected mitochondrial activity in eukaryotic cells. In conclusion, a 25-amino-acid peptide (LL7-31) displayed both strong antimicrobial and antibiofilm activities. The peptide was even active on cells within a preformed biofilm and had reduced toxicity toward eukaryotic cells. Our results also suggest the contribution of secondary structures ( -helix) to the activity of the peptides on biofilms.Item Biophysics of biofilm infection(2014-01) Stewart, Philip S.This article examines a likely basis of the tenacity of biofilm infections that has received relatively little attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength exceeds the external applied stress. Mechanical failure of the biofilm and release of planktonic microbial cells is also important in vivo because it can result in dissemination of infection. The fundamental criterion for detachment and dissemination is that the applied stress exceeds the biofilm failure strength. The apparent contradiction for a biofilm to both persist and disseminate is resolved by recognizing that biofilm material properties are inherently heterogeneous. There are also mechanical aspects to the ways that infectious biofilms evade leukocyte phagocytosis. The possibility of alternative therapies for treating biofilm infections that work by reducing biofilm cohesion could (1) allow prevailing hydrodynamic shear to remove biofilm, (2) increase the efficacy of designed interventions for removing biofilms, (3) enable phagocytic engulfment of softened biofilm aggregates, and (4) improve phagocyte mobility and access to biofilm.Item Antimicrobial Tolerance in Biofilms(2015-06) Stewart, Philip S.Tolerance to antimicrobial agents is a common feature of microbial biofilm formation ( 1 – 7 ). Table 1 presents a few examples of biofilm tolerance to biocides and antiseptics, and Table 2 summarizes some examples of antibiotic tolerance in biofilms. Neither of these listings is comprehensive, but these two data sets can be analyzed to gain insight into the factors that influence biofilm tolerance. The examples have been selected to illustrate the wide variety of microbial species, growth environments, and antimicrobial chemistries for which biofilm reduced susceptibility has been reported. The short list in Table 1 encompasses studies designed to mimic biofilms in dental plaque, hot tubs, paper mills, drinking water, household drains, urinary catheters, food processing plants, cooling water systems, and hospitals. These examples employ a range of individual and mixed species biofilms and diverse biocidal chemistries including halogens, phenolics, quaternary ammonium compounds, aldehydes, a plant essential oil, and peroxides. The studies captured in Table 2 cover 19 antibiotics and 9 organisms that include aerobic bacteria, strict anaerobes, and a fungus.