Scholarly Work - Center for Biofilm Engineering
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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 Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single-and dual-species cultures(2011-10) Behnke, S.; Parker, Albert E.; Woodall, Dawn; Camper, Anne K.Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.Item Antimicrobial penetration and efficacy in an in vitro oral biofilm model(2011-05) Corbin, A.; Pitts, Betsey; Parker, Albert E.; Stewart, Philip S.The penetration and overall efficacy of six mouthrinse actives was evaluated by using an in vitro flow cell oral biofilm model. The technique involved preloading biofilm cells with a green fluorescent dye that leaked out as the cells were permeabilized by a treatment. The loss of green color, and of biomass, was observed by time-lapse microscopy during 60 min of treatment under continuous flow conditions. The six actives analyzed were ethanol, sodium lauryl sulfate, triclosan, chlorhexidine digluconate (CHX), cetylpyridinium chloride, and nisin. Each of these agents effected loss of green fluorescence throughout biofilm cell clusters, with faster action at the edge of a cell cluster and slower action in the cluster center. The time to reach half of the initial fluorescent intensity at the center of a cell cluster, which can be viewed as a combined penetration and biological action time, ranged from 0.6 to 19 min for the various agents. These times are much longer than the predicted penetration time based on diffusion alone, suggesting that anti-biofilm action was controlled more by the biological action time than by the penetration time of the active. None of the agents tested caused any removal of the biofilm. The extent of fluorescence loss after 1 h of exposure to an active ranged from 87 to 99.5%, with CHX being the most effective. The extent of fluorescence loss in vitro, but not penetration and action time, correlated well with the relative efficacy data from published clinical trials.Item Performance of the AOAC use-dilution method with targeted modifications: Collaborative study(2012-11) Tomasino, S. F.; Parker, Albert E.; Hamilton, Martin A.; Hamilton, G. C.The U.S. Environmental Protection Agency (EPA), in collaboration with an industry work group, spearheaded a collaborative study designed to further enhance the AOAC use-dilution method (UDM). Based on feedback from laboratories that routinely conduct the UDM, improvements to the test culture preparation steps were prioritized. A set of modifications, largely based on culturing the test microbes on agar as specified in the AOAC hard surface carrier test method, were evaluated in a five-laboratory trial. The modifications targeted the preparation of the Pseudomonas aeruginosa test culture due to the difficulty in separating the pellicle from the broth in the current UDM. The proposed modifications (i.e., the modified UDM) were compared to the current UDM methodology for P. aeruginosa and Staphylococcus aureus. Salmonella choleraesuis was not included in the study. The goal was to determine if the modifications reduced method variability. Three efficacy response variables were statistically analyzed: the number of positive carriers, the log reduction, and the pass/fail outcome. The scope of the collaborative study was limited to testing one liquid disinfectant (an EPA-registered quaternary ammonium product) at two levels of presumed product efficacies, high and low. Test conditions included use of 400 ppm hard water as the product diluent and a 5% organic soil load (horse serum) added to the inoculum. Unfortunately, the study failed to support the adoption of the major modification (use of an agar-based approach to grow the test cultures) based on an analysis of method's variability. The repeatability and reproducibility standard deviations for the modified method were equal to or greater than those for the current method across the various test variables. However, the authors propose retaining the frozen stock preparation step of the modified method, and based on the statistical equivalency of the control log densities, support its adoption as a procedural change to the current UDM. The current UDM displayed acceptable responsiveness to changes in product efficacy; acceptable repeatability across multiple tests in each laboratory for the control counts and log reductions; and acceptable reproducibility across multiple laboratories for the control log density values and log reductions. Although the data do not support the adoption of all modifications, the UDM collaborative study data are valuable for assessing sources of method variability and a reassessment of the performance standard for the UDM.Item Direct electric current treatment under physiologic saline conditions kills Staphylococcus epidermidis biofilms via electrolytic generation of hypochlorous acid(2013-02) Sandvik, Elizabeth L.; McLeod, Bruce R.; Parker, Albert E.; Stewart, Philip S.The purpose of this study was to investigate the mechanism by which a direct electrical current reduced the viability of Staphylococcus epidermidis biofilms in conjunction with ciprofloxacin at physiologic saline conditions meant to approximate those in an infected artificial joint. Biofilms grown in CDC biofilm reactors were exposed to current for 24 hours in 1/10th strength tryptic soy broth containing 9 g/L total NaCl. Dose-dependent log reductions up to 6.7 log10 CFU/cm2 were observed with the application of direct current at all four levels (0.7 to 1.8 mA/cm2) both in the presence and absence of ciprofloxacin. There were no significant differences in log reductions for wells with ciprofloxacin compared to those without at the same current levels. When current exposures were repeated without biofilm or organics in the medium, significant generation of free chlorine was measured. Free chlorine doses equivalent to the 24-hour endpoint concentration for each current level were shown to mimic killing achieved by current application. Current exposure (1.8 mA/cm2) in medium lacking chloride and amended with sulfate, nitrate, or phosphate as alternative electrolytes produced diminished kills of 3, 2, and 0 log reduction, respectively. Direct current also killed Pseudomonas aeruginosa biofilms when NaCl was present. Together these results indicate that electrolysis reactions generating hypochlorous acid from chloride are likely a main contributor to the efficacy of direct current application. A physiologically relevant NaCl concentration is thus a critical parameter in experimental design if direct current is to be investigated for in vivo medical applications.Item Procedural revision to the AOAC germicidal spray products as disinfectants test method: Establishment of minimum and maximum log density values for test microbes on inoculated carriers(2013-06) Pines, R. M.; Tomasino, S. F.; Cottrill, M. P.; Hamilton, G. C.; Parker, Albert E.The AOAC Germicidal Spray Products as Disinfectants test method (AOAC Official Method 961.02) is used to measure the efficacy of spray products on hard inanimate surfaces; however, the method does not provide procedures to determine the population of the test microbe on inoculated glass slide carriers (e.g., carrier counts reported as CFU/carrier). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this issue and, based on carrier count data collected by four laboratories from 2000 to 2010, proposes a specific range for the mean log density per carrier as a requirement. Laboratory-based carrier count data were collected concurrently with 116 Method 961.02 efficacy tests conducted on spray products bearing claims against Pseudomonas aeruginosa and Staphylococcus aureus. For many of the tests a soil load (SL) was added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 696 carriers. All but two of the 116 mean log densities were at least 5.0 (a geometric mean of 1.0 × 105 CFU/carrier). Across the four combinations of microbes and SL treatments, the mean TestLD (mean log density across all enumerated carriers in a test) ranged from approximately 6.0 (a geometric mean of 0.9 × 106 CFU/carrier) to 6.3 (a geometric mean of 2.0 × 106 CFU/carrier). Across all microbes and SL treatments, the mean log density (±SEM) was 6.2 (±0.07) per carrier (a geometric mean of 1.5 × 106 CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.32) and reproducibility (0.34). The proposed requirement for S. aureus tests and P. aeruginosa tests is a mean log density (across six carriers) between 5.0 and 6.5. A separate 2009 study at three laboratories was conducted to evaluate the persistence of P. aeruginosa, S. aureus, and Salmonella enterica on glass carriers. Based on the persistence data, a 2 h use period is proposed for using the inoculated carriers post drying. The persistence data set was also used to assess the carrier counts for S. enterica. The carrier counts were approximately one log lower for S. enterica compared to S. aureus and P. aeruginosa; a range of 4.0 to 5.5 logs is proposed as a requirement for S. enterica tests.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 Health care worker hand contamination at critical moments in outpatient care settings(2016-11) Bingham, J.; Abell, G.; Kienast, L.; Lerner, L.; Matuschek, B.; Mullins, W.; Parker, Albert E.; Reynolds, N.; Salisbury, D.; Seidel, J.; Young, Elizabeth; Kirk, J.BACKGROUND: The delivery of health care in outpatient settings has steadily increased over the past 40 years. The risk of infection in these settings is considered to be low. However, the increasing severity of illness and complexity of care in outpatient settings creates a need to reexamine the transmission of pathogens in this setting. MATERIALS AND METHODS: Seventeen health care workers from 4 wound care facilities were sampled during 46 patient care encounters to determine the presence of health care-associated pathogens (ie, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, multidrug-resistant Acinetobacter species, and Clostridium difficile) on their hands at key moments of care. RESULTS: Health care workers acquired at least 1 pathogen on their hands during 28.3% of all patient care encounters. Hands sampled before a clean or aseptic procedure and hands sampled after body fluid exposure risk were each contaminated in 17.4% of instances. Hand contamination occurred in 19.6% of instances where health care workers wore gloves during care compared with 14.6% when health care workers were ungloved. CONCLUSIONS: Contamination of health care workers' hands presents a significant risk of pathogen transmission in outpatient settings. Gloving education, hand hygiene solutions at the point of care, and hand hygiene surveillance are important solutions for reducing transmission of pathogenic organisms.Item Ruggedness and reproducibility of the MBEC biofilm disinfectant efficacy test(2014-07) Parker, Albert E.; Walker, Diane K.; Goeres, Darla M.; Allan, N.; Olson, M. E.; Omar, A.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.Item Surface micropattern resists bacterial contamination transferred by healthcare practitioners(2014-12) Mann, Ethan E.; Mettetal, M. Ryan; May, Rhea M.; Drinker, M. C.; Stevenson, B. C.; Baiamonte, V. L.; Marso, J. M.; Dannemiller, E. A.; Parker, Albert E.; Reddy, Shravanthi T.; Sande, M. K.Environmental contamination contributes to an estimated 20-40% of all hospitalacquiredinfections (HAI). Infection control practices continue to improve, butmultipronged approaches are necessary to fully combat the diversity of nosocomialpathogens and emerging multidrug resistant organisms. The Sharkletâ„¢ micropattern,inspired from the microtopography of shark skin, was recently shown to significantlyreduce surface contamination but has not been evaluated in a clinical setting. Thefocus of this study was the transfer of bacteria onto micropatterned surfaces comparedto unpatterned surfaces in a clinical simulation environment involving healthcarepractitioners. Physician volunteers were recruited to participate in an emergencymedicine scenario involving a contact-precaution patient with an acute pulmonaryembolism. Prior to scenario initiation, Staphylococcus aureus was inoculated onto theleg of a simulation mannequin and fresh micropatterned and unpatterned surfacefilms were placed on a code cart, cardiac defibrillator shock button, and epinephrinemedication vial. Six physicians interacted with micropatterned surfaces and fivephysicians interacted with unpatterned surfaces in separate scenarios. Bacterial loadloss from the first contact location (control film over the femoral pulse) to subsequentunpatterned or micropatterned surface test locations was quantified as a log reduction(LR) for each surface type.The code cart, cardiac defibrillator button, and medication vial locations withmicropatterned surfaces resulted in LRs that were larger than the unpatternedLRs by 0.64 (p=0.146), 1.14 (p=0.023), and 0.58 (p=0.083) respectively for eachlocation. The geometric mean CFU/RODAC at the first control surface touched at thefemoral pulse pads ranged from 175-250 CFU/RODAC (95% confidence interval).Thus, the micropatterned LRs were consistently greater than the unpatterned LRs,substantiating the micropattern-dependent reduction of microorganism transfer.Principal component analysis showed that the LRs for the code cart and the cardiacdefibrillator button highly covaried. Thus, a single mean LR was calculated fromthese two locations for each surface type; 5.4 times more bacteria attached to theunpatterned surfaces compared to the micropatterned surfaces (p = 0.058). Thesimulated clinical scenario involving healthcare practitioners demonstrated that themicropatterned surface reduced the transfer of bacterial contamination based onthe larger LRs for the micropatterned surface compared to control surfaces. Furtherinvestigation in hospital rooms where patients are receiving care will ultimately revealthe capability of micropatterned surfaces to minimize the incidence of HAIs.