Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item Production of cell–cell signaling molecules by bacteria isolated from human chronic wounds(2010-05) Rickard, A. H.; Colacino, K. R.; Manton, K. M.; Morton, R. I.; Pulcini, Elinor D.; Pfeil, J.; Rhoads, Daniel D.; Wolcott, Randall D.; James, Garth A.Aim: To (i) identify chronic wound bacteria and to test their ability to produce acyl-homoserine-lactones (AHLs) and autoinducer-2 (AI-2) cell–cell signalling molecules and (ii) determine whether chronic wound debridement samples might contain these molecules. Methods and Results: Partial 16S rRNA gene sequencing revealed the identity of 46 chronic wound strains belonging to nine genera. Using bio-reporter assays, 69-6% of the chronic wound strains were inferred to produce AI-2, while 19-6% were inferred to produce AHL molecules. At least one strain from every genus, except those belonging to the genera Acinetobacter and Pseudomonas, were indicated to produce AI-2. Production of AI-2 in batch cultures was growth-phase dependent. Cross-feeding assays demonstrated that AHLs were produced by Acinetobacter spp., Pseudomonas aeruginosa and Serratia marcescens. Independent from studies of the bacterial species isolated from wounds, AHL and/or AI-2 signalling molecules were detected in 21 of 30 debridement samples of unknown microbial composition. Conclusion: Chronic wound bacteria produce cell–cell signalling molecules. Based on our findings, we hypothesize that resident species generally produce AI-2 molecules, and aggressive transient species associated with chronic wounds typically produce AHLs. Both these classes of cell–cell signals are indicated to be present in human chronic wounds. Significance and Impact of the Study: Interbacterial cell–cell signalling may be an important factor influencing wound development and if this is the case, the presence of AHLs and AI-2 could be used as a predictor of wound severity. Manipulation of cell–cell signalling may provide a novel strategy for improving 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 New horizons for cutaneous microbiology: the role of biofilms in dermatological disease(2011-09) Vlassova, Natalia; Han, Anne; Zenilman, Jonathan M.; James, Garth A.; Lazarus, Gerald S.Human skin is colonized by bacteria. The development of new genomic microbiological techniques has revealed that the bacterial ecology of human skin is far more complex than previously imagined and includes many fastidious or noncultivable bacterial species which are found on both normal and diseased skin. In nature, the predominant bacterial phenotype on epithelial surfaces is that of organisms organized within a biofilm. This contrasts with the widely held belief that bacteria are planktonic, i.e. free-floating single cells. Biofilms are sessile bacterial communities encased in an extracellular matrix that have a well-developed communication system and can regulate bacterial growth and metabolism, confer resistance to antimicrobials and to host inflammatory cells, and alter host metabolism. Biofilms have been observed on healthy skin and in a number of dermatological conditions, including some that were previously thought not to have an infectious aetiology. Here we review the concept of biofilms and their role in cutaneous health and disease.Item An in vitro model for the growth and analysis of chronic wound MRSA biofilms(2011-09) Agostinho, Alessandra; Hartman, A.; Lipp, C.; Parker, Albert E.; Stewart, Philip S.; James, Garth A.Aims: To develop an in vitro model (Colony/drip-flow reactor – C/DFR) for the growth and analysis of methicillin-resistant Staphylococcus aureus (MRSA) biofilms. Methods and Results: Using the C/DFR model, biofilms were grown on the top of polycarbonate filter membranes inoculated with a clinical isolate of MRSA, placed on absorbent pads in the DFR and harvested after 72 h. The biofilms varied from 256 to 308 µm in thickness with a repeatability standard deviation of 0·22. Testing of antimicrobial agents was also performed where C/DFR biofilms were grown in parallel with conventional colony biofilms. A saline solution (control), 1% silver sulfadiazine solution, and 0·25% Dakin’s solution were used to treat the biofilms for 15 min. Microscopic evaluation of biofilm morphology and thickness was conducted. The Dakins solution in both models produced statistically significantly higher log reductions than silver sulfadiazine treatment. Conclusions: The C/DFR biofilms were thick and repeatable and exhibited higher resistance to Dakins solution than the treated colony biofilms. Significance and Impact of the Study: The C/DFR can be used as a tool for examining complex biofilm physiology as well as for performing comparative experiments that test wound care products and novel antimicrobials.Item New methods for the detection of orthopedic and other biofilm infections(2011-03) Costerton, J. William; Post, J. C.; Ehrlich, Garth D.; Hu, Fen Z.; Kreft, R.; Nistico, L.; Kathju, S.; Stoodley, Paul; Hall-Stoodley, Luanne; Maale, G.; James, Garth A.; Sotereanos, N.; DeMeo, P.The detection and identification of bacteria present in natural and industrial ecosystems is now entirely based on molecular systems that detect microbial RNA or DNA. Culture methods were abandoned in the 1980s because direct observations showed that <1% of the bacteria in these systems grew on laboratory media. Culture methods comprise the backbone of the Food and Drug Administration-approved diagnostic systems used in hospital laboratories, with some molecular methods being approved for the detection of specific pathogens that are difficult to grow in vitro. In several medical specialties, the reaction to negative cultures in cases in which overt signs of infection clearly exist has produced a spreading skepticism concerning the sensitivity and accuracy of traditional culture methods.We summarize evidence from the field of orthopedic surgery, and from other medical specialties, that support the contention that culture techniques are especially insensitive and inaccurate in the detection of chronic biofilm infections. We examine the plethora of molecular techniques that could replace cultures in the diagnosis of bacterial diseases, and we identify the new Ibis technique that is based on base ratios (not base sequences), as the molecular system most likely to fulfill the requirements of routine diagnosis in orthopedic surgery.Item Combined treatment of Pseudomonas aeruginosa biofilm with lactoferrin and xylitol inhibits the ability of bacteria to respond to damage resulting from lactoferrin iron chelation(2011-04) Ammons, Mary Cloud B.; Ward, L. S.; Dowd, Scot E.; James, Garth A.With an ageing and ever more obese population, chronic wounds such as diabetic ulcers, pressure ulcers and venous leg ulcers are an increasingly relevant medical concern. Identification of bacterial biofilm contamination as a major contributor to non-healing wounds demands biofilm-targeted strategies to manage chronic wounds. Pseudomonas aeruginosa has been identified as a principal biofilm-forming opportunistic pathogen in chronic wounds. The innate immune molecule lactoferrin and the rare sugar alcohol xylitol have been demonstrated to be co-operatively efficacious against P. aeruginosa biofilms in vitro. Data presented here propose a model for the molecular mechanism behind this co-operative antimicrobial effect. Lactoferrin iron chelation was identified as the primary means by which lactoferrin destabilises the bacterial membrane. By microarray analysis, 183 differentially expressed genes of ≥1.5-fold difference were detected. Interestingly, differentially expressed transcripts included the operon encoding components of the pyochelin biosynthesis pathway. Furthermore, siderophore detection verified that xylitol is the component of this novel synergistic treatment that inhibits the ability of the bacteria to produce siderophores under conditions of iron restriction. The findings presented here demonstrate that whilst lactoferrin treatment of P. aeruginosa biofilms results in destabilisation of the bacterial cell membrane though iron chelation, combined treatment with lactoferrin and xylitol inhibits the ability of P. aeruginosa biofilms to respond to environmental iron restriction.Item Anti-biofilm efficacy of a lactoferrin/xylitol wound hydrogel used in combination with silver wound dressings(2011-04) Ammons, Mary Cloud B.; Ward, L. S.; James, Garth A.With an epidemic increase in obesity combined with an ageing population, chronic wounds such as diabetic foot ulcers, pressure ulcers and venous leg ulcers are an increasing clinical concern. Recent studies have shown that bacterial biofilms are a major contributor to wound bioburden and interfere with the normal wound healing process; therefore, rational design of wound therapies should include analysis of anti-biofilm characteristics. Studies using the combined treatment of bacterial biofilms with the innate immune molecule lactoferrin and the rare sugar-alcohol xylitol have demonstrated an antimicrobial capacity against a clinical wound isolate.Studies presented here used a colony-drip-flow reactor biofilm model to assess the anti-biofilm efficacy of a lactoferrin/xylitol hydrogel used in combination with commercially available silver-based wound dressings. Log reductions in biofilm viability are compared with a commercially available wound hydrogel used in combination with the silver-based wound dressings. For both a single species biofilm and a dual species biofilm, the lactoferrin/xylitol hydrogel in combination with the silver wound dressing Acticoatâ„¢ had a statistically significant reduction in biofilm viability relative to the commercially available wound hydrogel. This study also demonstrated a statistical interaction between the lactoferrin/xylitol hydrogel and the silver wound dressing.Item 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.Item An in vitro comparison of intraluminal biofilm bacteria transfer of three peripheral intravenous valved blood control catheters(2013-06) Ryder, M.; James, Garth A.; Pulcini, Elinor D.; Parker, Albert E.The insertion of peripheral intravenous catheters (PIVC) is the most common invasive procedure performed by nurses. The new generation of PIVCs developed to reduce blood exposure during insertion utilizes additional internal components within the catheter hub. These components increase the internal surface area that is thought to increase biofilm formation and subsequent transfer of bacteria into the bloodstream. This raises concern for increased risk of bloodstream infection. The purpose of the this study is to compare biofilm formation and bacterial transfer rate between 3 valved blood control PIVCs in a clinically simulated in vitro model.Item 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.