Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case, resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 10–15 doi:10.1038/sj.jim.7000256 Received 29 October 2001/ Accepted in revised form 18 March 2002     

Evaluation of a model biofilm for the ranking of biocide performance against sulphate-reducing bacteria

A model artificial biofilm was developed and evaluated for ranking the performance of biocides for application in oil production pipelines. The biofilm consisted of an alginate gel matrix into which were incorporated bacteria, scrapings from the inner surfaces of oil production pipelines and some crude oil.
The viability and sulphide-respiration rates of sulphate-reducing bacteria (SRB) within freshly-prepared artificial biofilm remained largely unchanged during a 2-week storage period. Furthermore, storage of the model biofilm did not alter the susceptibility of the incorporated SRB to a biocide. These findings showed that artificial biofilm may be produced in advance of a biocide assessment study and stored for at least 2 weeks over the course of the study without the model system undergoing changes which affected the relative performance of the biocides assessed. Artificial biofilms were found to be more resistant to biocides than planktonic bacteria and the addition of oil pipeline scrapings and crude oil to the artificial biofilm was found to increase further the resistance of biofilm to biocides.     

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.     

Modelling biofilm‐induced formation damage and biocide treatment in subsurface geosystems

Biofilm growth in subsurface porous media, and its treatment with biocides (antimicrobial agents), involves a complex interaction of biogeochemical processes which provide non‐trivial mathematical modelling challenges. Although there are literature reports of mathematical models to evaluate biofilm tolerance to biocides, none of these models have investigated biocide treatment of biofilms growing in interconnected porous media with flow. In this paper, we present a numerical investigation using a pore network model of biofilm growth, formation damage and biocide treatment. The model includes three phases (aqueous, adsorbed biofilm, and solid matrix), a single growth‐limiting nutrient and a single biocide dissolved in the water. Biofilm is assumed to contain a single species of microbe, in which each cell can be a viable persister, a viable non‐persister, or non‐viable (dead). Persisters describe small subpopulation of cells which are tolerant to biocide treatment. Biofilm tolerance to biocide treatment is regulated by persister cells and includes ‘innate’ and ‘biocide‐induced’ factors. Simulations demonstrate that biofilm tolerance to biocides can increase with biofilm maturity, and that biocide treatment alone does not reverse biofilm‐induced formation damage. Also, a successful application of biological permeability conformance treatment involving geologic layers with flow communication is more complicated than simply engineering the attachment of biofilm‐forming cells at desired sites.     

Reduced susceptibility of thin Pseudomonas aeruginosa biofilms to hydrogen peroxide and monochloramine

Pseudomonas aeruginosa attached to alginate gel beads in sparse, thin biofilms exhibited reduced susceptibility to monochloramine and hydrogen peroxide compared with planktonic cells of the same micro-organism. Disinfection rate coefficients for planktonic bacteria averaged 0.551 mg(-1)min(-1) for monochloramine and 3.1 x 10(-4)l mg(-1) min(-1) for hydrogen peroxide. The corresponding values for 24-h-old biofilm cells were 0.291 mg min(-1) and 9.2 x 10(-5) 1 mg(-1) min(-1) for monochloramine and hydrogen peroxide, respectively. Several pieces of evidence support the interpretation that the reduced susceptibility of biofilm was not due simply to inadequate delivery of the antimicrobial agent to the local environment of the attached cells. No correlation between biofilm susceptibility and biofilm initial areal cell density was observed. Rapid delivery of hydrogen peroxide to the attachment surface, and subsequently to the interior, of the alginate gel beads was visualized by a direct experimental technique. Theoretical analysis of unsteady diffusion and diffusion reaction interactions also argued against any significant delay or barrier to antimicrobial or oxygen delivery. It was hypothesized that new genes are expressed when bacteria attach to a surface and begin to form a biofilm and that some of the resulting gene products reduce the susceptibility of the cell to antimicrobial agents including oxidative biocides such as monochloramine and hydrogen peroxide.     

Modeling of Biocide Action Against Biofilm

We consider the mathematical model of dynamic antimicrobial action against bacterial biofilms. A mixture model is used in which the biofilm consisting of live and dead bacteria is modeled as one fluid component, while the solvent containing biocide is modeled as the other, and each component is represented by its volume fraction. The whole system is assumed to be an incompressible fluid and the velocity is governed by the Navier-Stokes equation. Biocide kills the live bacteria and its transport is governed by an advection-reaction-diffusion equation. Certain biocide also weakens the mechanical cohesiveness of the biofilm and results in biofilm removal under the shear stress of the external flow. Spatial and temporal patterns of antimicrobial action of three different biocides are considered and numerical simulation results by finite difference method are presented.     

The ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.     

Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine

The role of two sigma factors, AlgT and RpoS, in mediating Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine was investigated. Two knock out mutant strains, SS24 (rpoS-) and PAO6852 (algT-), were compared with a wild type, PAO1, in their susceptibility to monochloramine and hydrogen peroxide. When grown as biofilms on alginate gel beads (mean untreated areal cell density 3.7 +/- 0.27 log cfu cm-2) or on glass slides (mean untreated areal cell density 7.6 +/- 0.9 log cfu cm-2), wild type bacteria exhibited reduced susceptibility to both antimicrobial agents in comparison with suspended cells. On alginate gel beads, all strains were equally resistant to monochloramine. rpoS- and algT- gel bead biofilms of 24-hour-old were more susceptible to hydrogen peroxide disinfection than were biofilms formed by PAO1. Biofilm disinfection rate coefficients for the two mutant strains were statistically indistinguishable from planktonic disinfection rate coefficients, indicating complete loss of biofilm resistance. While 48-hour-old algT- biofilm cells became resistant to hydrogen peroxide, 48-hour-old rpoS- biofilm cells remained highly susceptible. With the thicker biofilms formed on glass coupons, all strains were equally resistant to both hydrogen peroxide and monochloramine. It is concluded that while RpoS and AlgT may play a transient role in protecting thin biofilms from hydrogen peroxide, these sigma factors do not mediate resistance to monochloramine and do not contribute significantly to the hydrogen peroxide resistance of thick biofilms.     

Efficacy of biocides used in the modern food industry to control salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

Artificial biofilm model – a useful tool for biofilm research

For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50–500 μm diameter) and porous beads (260 μm mean diameter) containing pores with diameters from 10 to 80 μm (28 μm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties. Received: 21 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

Efficacy of biocides on laboratory-generated Legionella biofilms

Results of biocide efficacy testing on laboratory-generated microbial biofilms containing Legionella bozemanii is presented. These show that chlorine is effective at recommended concentrations in cooling towers; at least 48 h contact between free chlorine and the biofilm is necessary. Of five commercial biocides tested, those containing isothiazolinones and dibromonitrilo-proprionamide were most effective against sessile L. bozemanii.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Influence of starvation, surface attachment and biofilm growth on the biocide susceptibility of the biodeteriogenic yeast Aureobasidium pullulans

AIM: To investigate the effect of starvation, surface attachment and growth in a biofilm on the susceptibility of Aureobasidium pullulans to the biocides 2-n-octyl-4-isothiazolin-3-one (OIT) and sodium hypochlorite (NaOCl). METHODS AND RESULTS: Fluorescence loss from a green fluorescent protein (GFP)-transformed strain was used to monitor real-time loss in viability as previously described in situ in 96-well plates. Exponential phase, yeast-like (YL) cells were settled in the bottom of the wells as a low-density monolayer (LDM) and were susceptible to all biocide concentrations (25-100 mug ml(-1)). The exponential phase YL cells were either starved for 48 h in suspension or starved for 48 h as LDMs in the wells. Starvation in both cases led to a small reduction in susceptibility to the biocides. In contrast, 48-h biofilms grown in malt extract broth showed an apparent lack of susceptibility to 25 and 50 mug ml(-1) OIT and to 25-100 mug ml(-1) NaOCl. However, when the OIT concentration was increased to compensate for the higher cell density in the biofilm, the biofilms were found to be equally susceptible to the LDM. CONCLUSIONS: Starvation of A. pullulans YL cells either in suspension or as attached LDM resulted in a decrease in susceptibility to low concentrations of both OIT and NaOCl while the apparent reduced susceptibility of mature biofilms was due to the increase in biofilm cell density rather than true biofilm resistance per se. SIGNIFICANCE AND IMPACT OF THE STUDY: Monitoring fluorescence loss from the GFP-transformed strain of A. pullulans can be used as a fast and reliable method for monitoring cell death in real time as a response to biocide and antimicrobial challenge.     

Effectiveness Factors and Conversion in a Biocatalytic Membrane Reactor

Analytical expressions of the effectiveness factor of a biocatalytic membrane reactor, and its asymptote as the Thiele modulus becomes large, are presented. The evaluation of the effectiveness factor is based on the solution of the governing equations for solute transport in the two regions of the reactor, i.e. the lumen and the matrix (with the biofilm immobilized in the matrix). The lumen solution accounts for both axial diffusion and radial convective flow, while the matrix solution is based on Robin-type boundary conditions. The effectiveness factor is shown to be a function of the Thiele modulus, the partition coefficient, the Sherwood number, the Peclet number, and membrane thickness. Three regions of Thiele moduli are defined in the effectiveness factor graphs. These correspond with reaction rate limited, internal-diffusion limited, and external mass transfer limited solute transport. Radial convective flows were shown to only improve the effectiveness factor in the region of internal diffusion limitation. The assumption of first order kinetics is shown to be applicable only in the Thiele modulus regions of internal and external mass transfer limitation. An iteration scheme is also presented for estimating the effectiveness factor when the solute fractional conversion is known. The model is validated with experimental data from a membrane gradostat reactor immobilised with Phanerochaete chrysosporium for the production of lignin and manganese peroxidases. The developed model and experimental data allow for the determination of the Thiele modulus at which the effectiveness factor and fractional conversion are optimal.     

Comparative susceptibility of resident and transient hand bacteria to para-chloro-meta-xylenol and triclosan

AIMS: To determine the susceptibility of planktonic and biofilm-grown strains of resident and transient skin bacteria to the liquid hand soap biocides para-chloro-meta-xylenol (PCMX) and triclosan. METHODS AND RESULTS: Freshly isolated hand bacteria were identified by partial 16S rRNA gene sequencing. Two resident and three transient strains, as well as four exogenous potential transient strains, were selected for biocide susceptibility testing. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of planktonic cells were determined. Resident and transient strains showed a range of susceptibilities to both biocides (PCMX, MIC 12.5-200 mg x l(-1), MBC 100-400 mg x l(-1); triclosan, MIC 0.6- > 40 mg x l(-1), MBC 1.3- > 40 mg x l(-1)). Strains were attached to polystyrene plates for 65 h in 96-well microtitre plates and challenged with biocide to determine the biofilm inhibitory concentration and biofilm eradicating concentration. For all strains tested, biofilms were two- to eightfold less susceptible than planktonic cells to PCMX. CONCLUSIONS: Very few transients were detected on the hand. Transients were not more sensitive than residents to the biocides and susceptibility to PCMX and triclosan was strain dependent. Biofilm-grown strains were less susceptible to PCMX than planktonic cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides increased knowledge about the susceptibility of skin bacteria to biocides present in typical liquid antibacterial hand soaps and suggests that the concentration of biocide employed in such products is in excess of that required to kill the low numbers of transient bacteria typically found on skin.     

Modeling biocide action against biofilms

A phenomenological model of biocide action against microbial biofilms was derived. Processes incorporated in the model include bulk flow in and out of a well-mixed reactor, transport of dissolved species into the biofilm, substrate consumption by bacterial metabolism, bacterial growth, advection of cell mass within the biofilm, cell detachment from the biofilm, cell death, and biocide concentration-dependent disinfection. Simulations were performed to analyze the general behavior of the model and to perform preliminary sensitivity analysis to identify key input parameters. The model captured several general features of antimicrobial agent action against biofilms that have been observed widely by experimenters and practitioners. These included (1) rapid disinfection followed by biofilm regrowth, (2) slower detachment than disinfection, and (3) reduced susceptibility of microorganisms in biofilms. The results support the plausibility of a mechanism of biofilm resistance in which the biocide is neutralized by reaction with biofilm constituents, leading to a reduction in the bulk biocide concentration and, more significantly, biocide concentration gradients within the biofilm. Sensitivity experiments and analyses identified which input parameters influence key response variables. Each of three response variables was sensitive to each of the five input parameters, but they were most sensitive to the initial biofilm thickness and next most sensitive to the biocide disinfection rate coefficient. Statistical regression modeling produced simple equations for approximating the response variables for situations within the range of conditions covered by the sensitivity experiment. The model should be useful as a tool for studying alternative biocide control strategies. For example, the simulations suggested that a good interval between pulses of biocide is the time to minimum thickness. (c) 1996 John Wiley & Sons, Inc.