Browsing by Author "Pasmore, M."

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The application of biofilm science to the study and control of bacterial infections
(2003-11) Costerton, J. William; Veeh, Richard Harold; Shirtliff, Mark E.; Pasmore, M.; Post, C.; Ehrlich, Garth D.
Unequivocal direct observations have established that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms. The diagnostic and therapeutic strategies that have served us so well in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases. We discuss the potential benefits of the application of the new methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases.
Biofilm removal from silicone tubing: an assessment of the efficacy of dialysis machine decontamination procedures using an in vitro model
(2003-01) Marion-Ferey, Karine; Pasmore, M.; Stoodley, Paul; Wilson, Suzanne; Husson, Gilles; Costerton, J. William
The aim of this study was to assess the efficacy of 21 decontamination procedures, for the removal of a multispecies biofilm. Experiments were performed on five-day-old biofilms grown inside silicone tubing, using a reactor system that mimics a dialysis machine. The treatments were tested on 5 cm tubing samples. Effects of treatment were measured using direct microscopy following staining. Bacterial viability and endotoxin removal were determined using conventional microbiological methods following biofilm detachment by scraping. The 21 procedures were classified into four groups based on the amount of biofilm removed. The most effective treatment was an acid pre-treatment, followed by use of a concentrated bleach solution. Acid pre-treatment removes calcium and magnesium carbonate crystals that are always found in dialysis biofilms. Treatments performed at high temperature did not increase the efficacy of biofilm removal. Most treatments caused at least a 105-fold reduction in bacterial viability with a few resulting in complete kill. Autoclaved and bleach-treated samples gave the best results for viability reduction, with both treatments providing an equally effective and complete kill. In addition, autoclaving led to a significant decrease in endotoxin level (removal of 99.99%).
Biofilms, bacterial signaling, and their ties to marine biology
(2003-07) Pasmore, M.; Costerton, J. William
Much of what is know about quorum sensing has come from the study of marine biology. The original description of the phenomenon was based on the study of marine bacteria and the luminescent pathway. More recently, aquatic organisms have been found to inhibit bacterial fouling of surfaces by blocking signaling pathways in the bacteria. These signaling effects have, over the last 5 years, been linked to biofilms. However, this correlation is not as straight forward as originally believed. Here, a brief overview of quorum sensing, and background on biofilms is provided, followed by a discussion of more recent work looking at the effects that environment may have on signal expression.
Biofilms, biomaterials and device-related infections
(2004) Costerton, J. William; Stoodley, Paul; Shirtliff, Mark E.; Pasmore, M.; Cook, Guy S.
Detection of staphylococcus aureus biofilm on tampons and menses components
(2003-08) Veeh, Richard Harold; Shirtliff, Mark E.; Petik, Jill R.; Flood, Janine A.; Davis, Catherine C.; Seymour, Jon L.; Hansmann, Melanie A.; Kerr, Kathy M.; Pasmore, M.; Costerton, J. William
Culturing has detected vaginal Staphylococcus aureus in 10%–20% of women. Because growth mode can affect virulence expression, this study examined S. aureus–biofilm occurrence in 44 paired—tampon and vaginal-wash—specimens from 18 prescreened women, using fluorescent in situ hybridization (FISH). All 44 specimens were also analyzed for S. aureus by standard culturing on mannitol salt agar, which produced positive results for 15 of the 44 specimens. FISH detected S. aureus cells in all 44 specimens, and S. aureus biofilm was observed in 37 of the 44 specimens. Independent confirmation of the presence of S. aureus in specimens from all 18 women was also obtained by amplification, via polymerase chain reaction, of an S. aureus–specific nuclease gene. The results of this study demonstrate that S. aureusbiofilm can form on tampons and menses components in vivo. Additionally, the prevalence of vaginal S. aureus carriage may be more prevalent than what is currently demonstrated by standard culturing techniques
Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid
(2005-07) Ren, Dacheng; Zuo, Rongjun; González Barrios, Andrés F.; Bedzyk, Laura A.; Eldridge, Gary R.; Pasmore, M.; Wood, Thomas K.
After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 µg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold whenadded upon inoculation and when added to a 24-h biofilm; however,ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa,Vibrio harveyi, and hepatocytes. Similarly, 10 µg of ursolicacid/ml inhibited biofilm formation by >87% for P. aeruginosain both complex and minimal medium and by 57% for V. harveyiin minimal medium. To investigate the mechanism of this nontoxicinhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 µg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 µg of ursolic acid/ml,respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism(cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid).
Effect of polymer surface properties on the reversibility of attachment of pseudomonas aeruginosa in the early stages of biofilm developmen
(2002-01) Pasmore, M.; Todd, Paul W.; Pfiefer, Blaine; Rhodes, Michael; Bowman, Christopher N.
Biofilm fouling is a common problem in industrial water and medical systems. Prevention of biofilm formation is often difficult because of the numerous potential attachment and adhesion mechanisms of bacteria, and therefore removal strategies are often necessary. Three surface properties, surface roughness, hydrophobicity (contact angle), and surface charge (zeta potential) were measured for several polymeric surfaces and related to specific biofilm characteristics. Biofilms of Pseudomonas aeruginosa were allowed to form on these surfaces for a period of 1-3 d, and the fraction of bacterial cells removed from each surface by exposure of biofilms to a standard shear stress was determined. Cells were most readily removed from the smoothest, most hydrophilic, neutral surfaces, with removal becoming more difficult at longer attachment times. This finding correlates directly with the finding that surfaces with these characteristics are most resistant to biofilm initiation. Therefore, it is possible to produce a surface from which bacteria can be more readily removed.
Endotoxin level measurement in hemodialysis biofilm using the whole blood assay
(2005-06) Marion-Ferey, Karine; Leid, Jeff G.; Bouvier, Ghislaine; Pasmore, M.; Husson, Gilles; Villagines, Rolland
Biofilms have been found on the inner surface of silicone tubing inside dialysis machines. Endotoxin releasing from those biofilms increases the bioincompatibility of dialysis liquids and leads to long-term inflammatory complications among dialysis patients. Endotoxin measurement is recommended for the control of dialysis liquids. This article describes the use of a new method, the Whole Blood Assay (WBA), for endotoxin quantification in dialysis biofilms. Biofilms were suspended in sterile water by scraping the tubing samples. Diluted blood samples from healthy donors were stimulated overnight with the contaminated suspension. Stimulated mononuclear cells released IL-1β in response to endotoxins. IL-1β level was then measured using an ultrasensitive ELISA method. We demonstrated a semilogarithmic model in which the optical densities measured after the ELISA assay increases linearly with the levels of endotoxin. This model allowed the determination of the amount of endotoxins in biofilm samples with a detection limit of 0.032 EU/mL. Most of the time, the amounts of endotoxin measured by the WBA were higher than those measured by the Limulus Amoebocyte Lysate (LAL) assay. This study suggested the presence of “endotoxin-like” compounds different from the lipopolysaccharides that are not detected by the LAL assay. We concluded that the LAL is necessary but insufficient to have a representative quantification of endotoxins that could be hazardous to patient health.
Host reactions to biomaterials and their evaluation
(2004) Anderson, Jacob Michael; Cook, Guy S.; Costerton, J. William; Hanson, S. R.; Hensten-Pettersen, Arne; Jacobsen, Nils; Johnson, Richard J.; Mitchell, Richard N.; Pasmore, M.; Schoen, Frederick J.; Shirtliff, Mark E.; Stoodley, Paul
Methods for biofilm analysis on silicone tubing of dialysis machines
(2003-07) Marion-Ferey, Karine; Enkiri, F.; Pasmore, M.; Husson, Gilles; Vilagines, Rolland
We describe an analytical protocol to study biofilms that develop inside silicone tubing of dialysis machines. This protocol has been set up with the help of a dynamic testing device reproducing dialysis conditions. The methodology includes direct microscopic observation, biofilm removal with an original mechanical biofilm scraper, quantitative analysis with culturable and total bacteria counting, and endotoxin level measurement using the LAL chromogenic kinetic assay. The analytical protocol has been assessed on 13 different clinical tubing samples. Most samples were contaminated by adherent cells and the thickest biofilms were found at the connection between the dialysis water distribution loop and the dialysis machine. The less contaminated samples had been removed from dialysis machines that were decontaminated with citric acid and autoclaving, showing the importance of the decontamination procedure for the prevention of biofilm development. This article shows that easy, rapid, reproducible, and economical methods are applicable for a routine analysis of biofilms that develop on dialysis systems and should be included in the regular control of the microbiological quality of dialysis liquids.
A microbiological and confocal microscopy study documenting a slime-producing Staphylococcus epidermidis isolated from a nylon corneal suture of a patient with antibiotic-resistant endophthalmitis
(2005-04) Nucci, Carlo; Artini, Marco; Pasmore, M.; Missiroli, Filippo; Costerton, J. William; Selan, Laura
Background: We describe a case of posttraumatic endophthalmitis unresponsive to systemic (amoxicillin+clavulanic acid and piperacillin/tazobactam), intra-ocular (vancomycin) and topical (ofloxacin, tetracycline and sulfametoxazole) antibiotic therapy. Microbiological and confocal microscopy studies of a nylon corneal suture revealed the presence of a slime-producing strain of Staphylococcus epidermidis.Methods: We describe the history and clinical presentation of a 77-year-old man in whom a high-grade posttraumatic endophthalmitis resolved only after the removal of a single nylon corneal suture. Microbiological investigations of the aqueous, vitreous and suture were performed, and the propensity of the suture-associated isolate to form biofilm was assessed using confocal microscopy. Results: A single stain of S. epidermidis was isolated from both aqueous and vitreous specimens and from the suture. The planktonic form of the isolate was susceptible in vitro to the antibiotics administered to the patient, but the strain was capable of forming biofilms and this phenotype showed resistance to high concentrations of the same antibiotics. Conclusions: The presence of a slime-producing strain of S. epidermidis should be considered in endophthalmitis that is unresponsive to specific antibiotic therapy, especially in cases in which an intra-ocular foreign body (e.g., a suture) is present.
A new procedure allowing the complete removal and prevention of hemodialysis biofilms
(2005-08) Marion-Ferey, Karine; Pasmore, M.; Freney, J.; Delawari, E.; Renaud, F.; Costerton, J. William; Traeger, J.
Most currently used disinfectants for dialysis machines have a good bactericidal efficacy on biofilms but leave dead cells on the surface. This contributes to the regrowth of biofilm and the release of pyrogens. A new anti-biofilm procedure consisting of sequential treatment combining enzymes and detergents is able to detach adherent cells. The efficacy of this procedure was assessed both in vitro and in reality. For in vitro studies, a biofilm model was set up. Studies were also performed in reality in a clinically used dialysis machine. Biofilm removal was first monitored by image analysis. Then the biomass was detached by scraping and quantified by plate counts and endotoxin level measurement. Treated samples were compared to untreated control samples. The procedure led to the complete detachment of the biomass, both in vitro and in the reality situation. The aim of this procedure is to replace or complete the usual disinfection methods for medical devices.
Predictive modeling for hot water inactivation of planktonic and biofilm-associatedSphingomonas parapaucimobilis to support hot water sanitization programs
(2016-06) Kaatz Wahlen, L.; Parker, Albert E.; Walker, Diane K.; Pasmore, M.; Sturman, Paul J.
Hot water sanitization is a common means to maintain microbial control in process equipment for industries where microorganisms can degrade product or cause safety issues. This study compared the hot water inactivation kinetics of planktonic and biofilm-associated Sphingomonas parapaucimobilis at temperatures relevant to sanitization processes used in the pharmaceutical industry, viz. 65, 70, 75, and 80°C. Biofilms exhibited greater resistance to hot water than the planktonic cells. Both linear and nonlinear statistical models were developed to predict the log reduction as a function of temperature and time. Nonlinear Michaelis-Menten modeling provided the best fit for the inactivation data. Using the model, predictions were calculated to determine the times at which specific log reductions are achieved. While ≥80°C is the most commonly cited temperature for hot water sanitization, the predictive modeling suggests that temperatures ≥75°C are also effective at inactivating planktonic and biofilm bacteria in timeframes appropriate for the pharmaceutical industry.