Statistical assessment of a laboratory method for growing biofilms

Abstract

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 log 10 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.

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…This system has been heavily used to identify potential anti-biofilm agents, by measuring the reduction in surface attached biomass on the sides of wells after treatment with potential therapeutic agents [51][52][53][54]. In addition to this, The Center for Disease Control (CDC) approved biofilm reactor [55][56][57], and drip flow reactors, have proved excellent for assessing biofilm formation on biological and non-biological materials [58][59][60]. These flow systems and microtitre assays are the workhorses of in vitro biofilm research, and have generated a phenomenal amount of data that has greatly expanded our knowledge about how bacteria attach and differentiate into mature biofilms in vitro.…”

“…Other differences include the liquid flowing over the surface. During in vitro biofilm growth, well-defined minimal medias are most often used [49,56,57,71,72]. In catheters, the liquids flowing over the surface could be urine, blood or other bodily fluids, which constitutes a very different growth environment for bacteria.…”

New trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches

“…The SOPs proposed for biofilm studies are specific for: the growth of reproducible mixed biofilms of P. aeruginosa (ATCC#700829), Pseudomonas fluorescens (ATCC# 700830) and Klebsiella pneumoniae (ATCC#700831), analyzed in terms of structure and viable cell counts using a flat-plate, open channel reactor (Jackson et al 2001); the growth of P. aeruginosa biofilm using the CDC biofilm reactor (CBR) system (Goeres et al 2005); the assessment of resistance of Staphylococcus aureus biofilm cells to disinfectants (Luppens et al 2002); and the optimized quantification of enterococci biofilms using microtitre-plates (Extremina et al 2011). Also, as a first step towards the establishment of a SOP for methods of analysis, several authors have studied and compared methods in use on biofilm related research for evaluating biofilm disinfectant efficacy with different fluid dynamics (CDC, drip flow and static biofilm reactor systems) (Buckingham-Meyer et al 2007), comparing methods for quantification of microbial biofilms grown in microtiter plates (crystal violet (CV), Syto9, fluorescein diacetate (FDA), resazurin, XTT and dimethyl methylene blue (DMMB) assays) (Peeters et al 2008), and checking the validity of the harvesting and disaggregating steps (Hamilton et al 2009).…”

Application of a Novel Tongue Resin Replication System for in vitro Biofilm Studies

“…Indeed, statistical analysis performed by Goeres [25] demonstrated that the CDC biofilm reactor system serves as a reliable experimental tool to study biofilm formation evolving from a wide range of organisms.…”