Bactericidal Efficacy of Oxidized Silver against Biofilms Formed by Curtobacterium flaccumfaciens pv. Flaccumfaciens
Authors : Michael W. Harding, Lyriam L. R. Marques, Nick Allan, Merle E. Olson, Brenton Buziak, Patricia Nadworny, Amin Omar, Ronald J. Howard, and Jie Feng
Abstract : Bacterial wilt is a re-emerging disease on dry bean and can affect many other crop species within the Fabaceae. The causal agent, Curtobacterium flaccumfaciens pv. flaccumfaciens (CFF), is a small, Gram-positive, rod-shaped bacterium that is seed-transmitted. Infections in the host become systemic, leading to wilting and economic loss. Clean seed programs and bactericidal seed treatments are two critical management tools. This study characterizes the efficacies of five bacteri¬cidal chemicals against CFF. It was hypothesized that this bacterium was capable of forming biofilms, and that the cells within biofilms would be more tolerant to bactericidal treatments. The minimum biocide eradi¬cation concentration assay protocol was used to grow CFF biofilms, expose the biofilms to bactericides, and enumerate survivors compared to a non-treated control (water). Streptomycin and oxysilver bisulfate had EC95 values at the lowest concentrations and are likely the best candidates for seed treatment products for controlling seed-borne bacterial wilt of bean. The results showed that CFF formed biofilms during at least two phases of the bacterial wilt disease cycle, and the biofilms were much more difficult to eradicate than their planktonic counterparts. Overall, biofilm formation by CFF is an important part of the bacterial wilt disease cycle in dry edible bean and antibiofilm bactericides such as streptomycin and oxysilver bisulfate may be best suited for use in disease management.
Keywords : bacterial, bactericide, bean, biofilm, EC50
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Anti-biofilm activity of two novel, borate based, bioactive glass wound dressings
Authors : Steven Jung, Ted Day, Tyler Boone, Brenton Buziak, and Amin Omar
Abstract : Bioactive glass was first developed in the late 1960’s as a compound that would facilitate bone re-growth. In more recent years, this technology has been used to promote wound healing through its ability to stimulate soft tissue growth, angiogenesis, reduce inflammation, and prevent infection. Chronically infected wounds, which result from biofilm formation, affect millions of patients in the Unites States each year and cost billions of dollars to treat. The present studies demonstrate exposure of preformed biofilms to bioactive glass, under simulated body conditions, resulted in significant reduction in bacterial load. Additionally, specific therapeutically active metal ions such as copper and zinc were added to a borate bioactive glass formulation through a process of ion doping and found to further enhance the anti-biofilm activity. Based on the present findings, the antibiofilm agents released by borate bioactive glasses may prove effective to eradicate the biofilm infections that prevent healing in patients with chronic wounds.
Keywords : Biofilm, Bioactive Glass, Minimum Biofilm Eradication Concentration, MBEC Assay, Chronic Wounds, Copper, Zinc, Borate Bioactive Glass
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Improved Methods for Treatment of Phytopathogenic Biofilms: Metallic Compounds as Anti-Bacterial Coatings and Fungicide Tank-Mix Partners
Authors : Michael Harding; Patricia Nadworny; Brenton Buziak; Amin Omar; Greg Daniels; Jie Feng
Abstract : Fungi and bacteria cause disease issues in cultivated plants world-wide. In most cases, the fungi and bacteria colonize plant tissues as biofilms, which can be very challenging to destroy or eradicate. In this experiment, we employed a novel (biofilm) approach to crop disease management by evaluating the efficacies of six fungicides, and four silver-based compounds, versus biofilms formed by fungi and bacteria, respectively. The aim was to identify combinations of fungicides and metallic cations that showed potential to improve the control of white mold (WM), caused by the ascomycete fungus Sclerotinia sclerotiorum, and to evaluate novel high valency silver compounds as seed coatings to prevent biofilm formation of four bacterial blight pathogens on dry bean seeds. Our results confirmed that mature fungal biofilms were recalcitrant to inactivation by fungicides. When metallic cations were added to the fungicides, their efficacies were improved. Some improvements were statistically significant, with one combination (fluazinam + Cu2+) showing a synergistic effect. Additionally, coatings with silver compounds could reduce bacterial blight biofilms on dry bean seeds and oxysilver nitrate was the most potent inhibitor of bacterial blight.
Keywords : Oxysilver nitrate; pentasilver hexaoxoiodate; silver; copper; white mold; bacterial blight; Sclerotinia sclerotiorum; Pseudomonas; Xanthomonas
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Microbial Biofilms and Chronic Wounds
Authors : Amin Omar, J. Barry Wright, Gregory Schultz, Robert Burrell, Patricia Nadworny
Abstract : Background is provided on biofilms, including their formation, tolerance mechanisms, structure, and morphology within the context of chronic wounds. The features of biofilms in chronic wounds are discussed in detail, as is the impact of biofilm on wound chronicity. Difficulties associated with the use of standard susceptibility tests (minimum inhibitory concentrations or MICs) to determine appropriate treatment regimens for, or develop new treatments for use in, chronic wounds are discussed, with alternate test methods specific to biofilms being recommended. Animal models appropriate for evaluating biofilm treatments are also described. Current and potential future therapies for treatment of biofilm-containing chronic wounds, including probiotic therapy, virulence attenuation, biofilm phenotype expression attenuation, immune response suppression, and aggressive debridement combined with antimicrobial dressings, are described
Keywords : quorum sensing; antibiotic tolerance; antibiotic resistance; exopolymer; inflammation; wound healing; MBEC (minimum biofilm eradication concentration)
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Antimicrobial efficacy validation using in vitro and in vivo testing methods
Authors : Amin Omar, and Patricia Nadworny
Abstract : This work explores the unique antibiofilm activity of pentasilver hexaoxoiodate (Ag5IO6). To test this activity, wound dressings were impregnated with Ag5IO6 and compared with various commercially available silver-containing dressings, as well as dressings containing chlorhexidine, iodine and polyhexamethylene biguanide (PHMB). The materials were tested against Pseudomonas eruginosa, Staphylococcus aureus and Candida albicans for their ability to prevent micro-organism adherence, eliminate planktonic micro-organisms and disrupt/eliminate mature biofilms generated using the MBEC™ assay within 24 h of microbial exposure. Only the Ag5IO6-containing dressings were able to prevent adherence and eliminate surrounding planktonic micro-organisms for all species tested for ≥28 days of elution with log reductions >4. Two other silver dressings succeeded against P. aeruginosa only after 28 elution days, whilst the PHMB dressing succeeded after 28 days of elution against C. albicans only. Ag5IO6-containing dressings were able to generate >4 log reductions against all biofilms tested. The only commercial dressings able to generate >4 log reductions against biofilms were iodine against P. aeruginosa and S. aureus, and PHMB against S. aureus. The Ag5IO6 dressings demonstrated complete kill (>4 log reduction) in a standard 30-min planktonic log reduction assay against all species. These results demonstrate that Ag5IO6 has superior activity to a number of antimicrobials, with broad-spectrum efficacy that includes long-term prevention of microbial adherence, rapid kill of planktonic micro-organisms, and the ability to disrupt and eliminate mature biofilms. Thus, Ag5IO6 may be a valuable antimicrobial agent for use in a number of medical device applications, including wound dressings, various catheters or implants.
Keywords : Anti-biofilm Silver periodate Infection Nanostructure Medical device Anti-adherence
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Can a Stool Management System Prevent Environmental Spread of Clostridium Difficile: A Comparative Trial
Authors : Mikel Gray, Amin Omar, Brenton Buziak
Abstract : PURPOSE: The purpose of this study was to compare contamination of the immediate environment with Clostridium difficile spores and vegetative cells from 2 stool management systems over a period of 30 days in a controlled laboratory setting. DESIGN: In vitro, comparison trial. METHODS: Two stool management systems were compared over a 30-day period in a controlled laboratory setting. Sixteen systems were filled with sterile loose canine stool inoculated with 10 6 colony-forming units (CFUs) per milliliter of C difficile; specially prepared culture media were used to detect C difficile contamination on various surfaces of the device and in the immediate environment. Containment bags were changed daily and devices were refilled with inoculated stool to more closely imitate use in the clinical setting. A dichotomous outcome variable (growth vs no growth) was used to analyze contamination on a daily basis via the generalized estimating equation; devices were also compared on days 3, 10, 20, and 30 by measuring CFUs per device surface. Logistic regression analysis was used to analyze growth over time. When observations showed no growth, the Cochran Mantel Haenszel test was used to compare study devices. RESULTS: Analysis revealed that 20.8% of anterior surfaces of the collection bags for device 1 were contaminated versus 83.9% of collection bags for device 2 ( P < .001). Comparison of the tubing/hub interface resulted in similar findings; 20.8% of device 1 group were contaminated versus 86.3% of device 2 group ( P < .001). Analysis of an absorbent pad placed under the device during daily changes found that 0.5% of device 1 were contaminated versus 38.1% of pads placed under device 2 ( P < .001). CONCLUSIONS: Findings from this in vitro study show that stool management systems can limit or prevent environmental contamination of C difficile. Results also reveal significant differences in the 2 systems tested; we hypothesize that these differences are attributable to
Keywords : Clostridium difficile, fecal incontinence , infectious diarrhea , stool management systems .
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Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test. Journal of Microbiological Methods
Authors : A.E. Parker, D. K. Walker, D.M. Goeres, N. Allan, M.E. Olson, A. Omar
Abstract : The MBEC™ Physiology & Genetics Assay recently became the first approved ASTM standardized biofilm disinfectant efficacy test method. This report summarizes the results of the standardization process using Pseudomonas aeruginosa biofilms. Initial ruggedness testing of the MBEC method suggests that the assay is rugged (i.e., insensitive) to small changes to the protocol with respect to 4 factors: incubation time of the bacteria (when varied from 16 to 18 h), treatment temperature (20–24 °C), sonication duration (25–35 min), and sonication power (130–480 W). In order to assess the repeatability of MBEC results across multiple tests in the same laboratory and the reproducibility across multiple labs, an 8-lab study was conducted in which 8 concentrations of each of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms using the MBEC method. The repeatability and reproducibility of the untreated control biofilms were acceptable, as indicated by small repeatability and reproducibility standard deviations (SD) (0.33 and 0.67 log10(CFU/mm2), respectively). The repeatability SDs of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. In addition, for each of the 3 disinfectant types considered, the assay was statistically significantly responsive to the increasing treatment concentrations.
Keywords : Biofilm, Ruggedness, Reproducibility
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In vitro evaluations of microbial biofilms and their responses to chemical disinfectants.
Authors : M.W. Harding, G.D. Daniels , R.J. Howard, L.L.R. Marques, N.D. Allan, A. Omar , M.E. Olson
Abstract : For many pathogenic microbes, biofilm formation is a critical component of the disease cycle and is required for pathogenicity or full virulence. Recent innovation in in vitro biofilm reactors has allowed high throughput evaluation and testing of chemical treatments against microbial biofilms. For example, the BESTTM Assay is a versatile and specialized biofilm reactor capable of culturing bacterial and fungal biofilms in a replicated, high throughput, multi-well format. The microorganism forms a biofilm on the surfaces of pegs extending into liquid media contained within the microtitre plate wells. Additionally, planktonic cells also grow within the media in each well which allows simultaneous growth of biofilms and planktonic cultures within the wells of each reactor. Comparisons and evaluations of microbial biofilms on different surface materials can also be performed. For example, the bacterial ring rot pathogen Clavibacter michiganensis subsp. sepedonicus was studied in the BESTTM Assay to determine the conditions that promote growth in the biofilm form, the sensitivities of biofilm and planktonic cells to biocides, and the effects of biocide concentrations, exposure times and substrate types on efficacy. Technologies such as the BESTTM Assay excel at efficacy testing of chemical and biological agents being screened or developed for control of plant diseases, including postharvest diseases. Plant pathology research, phenotype screening, evaluating and formulating biological control products and protocols often ignore the critical aspect of biofilm potential. As a result, biofilm biology remains uncharacterized or unconfirmed for many plant-pathogen interactions and for most biocontrol agents. Biofilm reactors, such as the BESTTM Assay, provide technology for characterizing and evaluating these important issues.
Keywords : biofilms, bacterial ring rot, BESTTM Assay, disinfection, biocide, efficacy
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