CDC Biofilm Reactor Research Articles

Observing the Biofilm Matrix ofStaphylococcus epidermidisATCC 35984 Grown Using the CDC Biofilm Reactor

Bacteria flourish in nearly every environment on earth. Contributing to their ability to grow in many esoteric locations is their development into a biofilm structure. In an effort to more accurately model the growth environment of biofilms in nature, a Center for Disease Control and Prevention (CDC) biofilm reactor has been developed that mimics nature-like shear forces and renewable nutrient sources. To date, there has been no confirmation by scanning electron microscopy (SEM) that mature biofilms develop on a surface when grown using the CDC biofilm reactor. Three different SEM methods were used to collect images of Staphylococcus epidermidis ATCC 35984 that was to be grown using the CDC biofilm reactor. In addition, two different fixative techniques were used in each of the imaging methods. Results indicated that after 48 hours of growth in the reactor, S. epidermidis ATCC 35984 does produce a significant network of matrix components and 3D mushroom- or pillar-like structures with signs of water channel development. In conclusion, S. epidermidis ATCC 35984 grown using the CDC biofilm reactor does appear to display signs of mature biofilm development. These results could be important for studies wherein mature biofilms are needed for in vitro and/or in vivo applications.

Validation of the CDC biofilm reactor as a dynamic model for assessment of encrustation formation on urological device materials

Contemporary medical science is reliant upon the rational selection and utilization of devices, and therefore, an increasing need has developed for in vitro systems aimed at replicating the conditions to which urological devices will be subjected to during their use in vivo. We report the development and validation of a novel continuous flow encrustation model based on the commercially available CDC biofilm reactor. Proteus mirabilis-induced encrustation formation on test biomaterial sections under varying experimental parameters was analyzed by X-ray diffraction, infrared- and Raman spectroscopy and by scanning electron microscopy. The model system produced encrusted deposits similar to those observed in archived clinical samples. Results obtained for the system are highly reproducible with encrustation being rapidly deposited on test biomaterial sections. This model will have utility in the rapid screening of encrustation behavior of biomaterials for use in urological applications.

PR52P TO ASSESS THE EFFECTIVENESS OF TOPICAL NEGATIVE PRESSURE (TNP) COMBINED WITH LOCAL INSTILLATION OF BIOCIDES AGAINST STAPHYLOCOCCUS EPIDERMIDIS AND PSEUDOMONAS AERUGINOSA BIOFILM IN VITRO

Introduction: S epidermidis is a recognised major biofilm pathogen and has been implicated in medical device contamination ranging from artificial hip prostheses to breast implants. Pseudomonas aeruginosa is a known pathogen in chronic wounds. Within biofilms, bacteria are enclosed in a polymeric matrix or EPS which cements the bacteria to each other and to the surface. Biofilms act to protect the bacteria from the environment increasing resistance to host immunity, antibiotics and biocides. More recently, we have shown that biofilm occurs on the surface of chronic wounds and that this may have aetiological implications. Method: Staph spp and pseudomonas spp biofilm were grown on glass coupons using the CDC biofilm reactor for 48 hrs and the coupons randomly inserted into the wound model and for 24 hrs subjected to: 1 No instillation plus TNP 2 0.1% w/v formulated hypocholorous acid instillation plus TNP 3 Saline instillation (no biocide) plus TNP 4 Betadine instillation plus TNP Betadine and saline instillations were for 30 minutes every 8, 4 and 2 hours per day in separate experiments. NVC instillation was for 3 minutes, every 8, 4 and 2 hours per day in separate experiments. Following treatment, the coupons were carefully extracted to avoid damaging the biofilms. The effect of TNP on the biofilms was then assessed by colony forming units and electron microscopy. Results: The results show that lower frequency of instillation did not have significant effect on bacterial load for both types of bacteria. Increase in frequency of instillations resulted in no growth of pseudomonas while increase in frequency of instillations resulted in a significant decrease in staph spp.

Assessing the antimicrobial activity of zinc oxide thin films using disk diffusion and biofilm reactor

The electronic and chemical properties of semiconductor materials may be useful in preventing growth of microorganisms. In this article, in vitro methods for assessing microbial growth on semiconductor materials will be presented. The structural and biological properties of silicon wafers coated with zinc oxide thin films were evaluated using atomic force microscopy, X-ray photoelectron spectroscopy, and MTT viability assay. The antimicrobial properties of zinc oxide thin films were established using disk diffusion and CDC Biofilm Reactor studies. Our results suggest that zinc oxide and other semiconductor materials may play a leading role in providing antimicrobial functionality to the next-generation medical devices.

Association and decontamination of Bacillus spores in a simulated drinking water system

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log 10 increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log 10 reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.

Comparative evaluation of biofilm disinfectant efficacy tests

Regulatory agencies are receiving registration applications for unprecedented, antibiofilm label claims for disinfectants. Reliable, practical, and relevant laboratory biofilm test methods are required to support such claims. This investigation describes the influence of fluid dynamics on the relevancy of a laboratory test. Several disinfectant formulations were tested using three different biofilm testing systems run side-by-side: the CDC biofilm reactor system that created turbulent flow (Reynolds number between 800 and 1850), the drip flow biofilm reactor system that created slow laminar flow (Reynolds number between 12 and 20), and the static biofilm system that involved no fluid flow. Each comparative experiment also included a dried surface carrier test and a dried biofilm test. All five disinfectant tests used glass coupons and followed the same steps for treatment, neutralization, viable cell counting, and calculating the log reduction (LR). Three different disinfectants, chlorine, a quaternary ammonium compound, and a phenolic, were each applied at two concentrations. Experiments were conducted separately with Pseudomonas aeruginosa and Staphylococcus aureus and every experiment was independently repeated. The results showed that biofilm grown in the CDC reactor produced the smallest LR, the static biofilm produced the largest LR, and biofilm grown in the drip flow reactor produced an intermediate LR. The differences were large enough to be of practical importance. The dried surface test often produced a significantly higher LR than the tests against hydrated or dried biofilm. The dried biofilm test produced LR values similar to those for the corresponding hydrated biofilm test. These results show that the efficacy of a disinfectant must be measured by using a laboratory method where biofilm is grown under fluid flow conditions similar to the environment where the disinfectant will be applied.

Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms

A common assay to measure yeast metabolic activity in biofilms is based on the reduction of the tetrazolium salt XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} to a colored formazan. However, a recent report, also confirmed by our own findings about the shortcomings of the chromogenic XTT assay, has prompted us to investigate alternative methods for yeast biomass quantification. To this end, two fluorogenic assays using fluorescein diacetate (FDA) and SYTO® 9 as well as the XTT assay were comparatively evaluated with regard to the linear range of Candida albicans and Candida parapsilosis cell number–response curves, precision and intra- and interspecies variability. Reading of fluorescence and absorbance was carried out in a multilabel microtiter plate reader. All three assays were adequate for the determination of planktonic yeast biomass, but the FDA and SYTO® 9 assays present practical advantages. When applied to the quantification of yeast biofilm biomass obtained in the CDC biofilm reactor, the FDA assay proved superior.

Statistical assessment of a laboratory method for growing biofilms

Microbial biofilms have been grown in laboratories using a variety of different approaches. A laboratory biofilm reactor system, called the CDC biofilm reactor (CBR) system, has been devised for growing biofilms under moderate to high fluid shear stress. The reactor incorporates 24 removable biofilm growth surfaces (coupons) for sampling and analysing the biofilm. Following preliminary experiments to verify the utility of the CBR system for growing biofilms of several clinically relevant organisms, a standard operating procedure for growing a Pseudomonas aeruginosa biofilm was created. This paper presents the results of a rigorous, intra-laboratory, statistical evaluation of the repeatability and ruggedness of that procedure as well as the results of the experiments with clinically relevant organisms. For the statistical evaluations, the outcome of interest was the density (c.f.u. cm(-2)) of viable P. aeruginosa. Replicate experiments were conducted to assess the repeatability of the log density outcome. The mean P. aeruginosa log10 density was 7.1, independent of the coupon position within the reactor. The repeatability standard deviation of the log density based on one coupon per experiment was 0.59. Analysis of variance showed that the variability of the log density was 53 % attributable to within-experiment sources and 47 % attributable to between-experiments sources. The ruggedness evaluation applied response-surface design and regression analysis techniques, similar to those often used for sensitivity analyses in other fields of science and engineering. This approach provided a quantitative description of ruggedness; specifically, the amount the log density was altered by small adjustments to four key operational factors--time allowed for initial surface colonization, temperature, nutrient concentration, and fluid shear stress on the biofilm. The small size of the regression coefficient associated with each operational factor showed that the method was rugged; that is, relatively insensitive to minor perturbations of the four factors. These results demonstrate that the CBR system is a reliable experimental tool for growing a standard biofilm in the laboratory and that it can be adapted to study several different micro-organisms.