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.     

Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.     

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.     

Analysis of biocide transport limitation in an artificial biofilm system

An alginate gel bead artificial biofilm system was used to assay biofilm susceptibility to four biocides and to analyse the extent to which each agent penetrated the biofilm. Chlorine, glutaraldehyde, an isothiazolone, and a quaternary ammonium compound were tested on alginate-entrapped Enterobacter aerogenes in gel beads ranging from 1·8 to 6 mm in diameter. Gel-entrapped bacteria were less susceptible to all four antimicrobial agents than were planktonic micro-organisms. The degree of kill measured in artificial biofilm gel beads depended on the size of the gel bead and the cell density at which it was loaded. Disinfection efficacy decreased as gel bead radius or cell density increased. The manifest dependence of biofilm disinfection efficacy on the physical properties of the artificial biofilm (radius and cell density) suggests the impingement of transport limitation of biocide transport into the biofilm. A previously developed theory of biocide reaction and diffusion in biofilm was tested by calculating an appropriate Thiele modulus. In accordance with the theory, the efficacy of all four biocides decreased, albeit noisily, as the Thiele modulus exceeded 1. This result demonstrates that transport limitation can impact antimicrobial performance against biofilms not only of oxidizing biocides but also of non-oxidizing agents.     

Approaches to prevention, removal and killing of biofilms

Biofilms contribute to hygiene problems in the food industry and in the medical field. Biofilms are diverse and due to the development of special phenotypes, biofilm organisms are not as susceptible to biocides as planktonic microorganisms. Biofilms may be prevented by regular disinfection. Since the attachment of microbes to surfaces and the development of biofilm phenotypes is a very fast process, it is, however, almost impossible, to prevent biofilm formation completely. The removal and killing of established biofilms requires harsh treatments, mostly using oxidising biocides. Depending on the nature of the biofilms, different biocides may be useful and the best biocide for a specific biofilm still has to be determined under practical conditions. Another approach is the prevention of biofilm formation by selection of materials that do not support the attachment of microorganisms. Some materials like glass and stainless steel show less biofilm formation than others. The ranking of materials, however, depends on the conditions, under which they are tested. A novel approach is biofilm inhibition by supplementation of systems with nutrients, to inhibit attachment. First results on inhibition of biofouling in reversed osmosis systems are presented.     

A green biocide enhancer for the treatment of sulfate-reducing bacteria (SRB) biofilms on carbon steel surfaces using glutaraldehyde

Generally speaking, a much higher concentration of biocide is needed to treat biofilms compared to the dosage used to for planktonic bacteria. With increasing restrictions of environmental regulations and safety concerns on large-scale biocide uses such as oil field applications, it is highly desirable to make more effective use of biocides. In this paper a green biocide enhancer ethylenediaminedisuccinate (EDDS) that is a biodegradable chelator, was found to enhance the efficacy of glutaraldehyde in its treatment of sulfate-reducing bacteria (SRB) biofilms. Experiments were carried out in 100 ml anaerobic vials with carbon steel coupons. The ATCC 14563 strain of Desulfovibrio desulfuricans was used. Biofilms on coupon surfaces were visualized using scanning electron microscopy (SEM). Experimental results showed that EDDS reduced the glutaraldehyde dosages considerably in the inhibition of SRB biofilm establishment and the treatment of established biofilms on carbon steel coupon surfaces.     

Control of biocorrosion using laboratory and field assessments

Biofouling and biocorrosion can be significant problems in oilfield water injection systems, despite extensive use of chemical biocides. This work set out to establish relevant testing and monitoring procedures to optimise microbiological control in these systems.A combination of laboratory based sessile biocide screening trials and onsite monitoring of biofilm bacteria was developed to ensure that an effective biocontrol programme was set up. It is important to use a mixed population of bacteria freshly isolated from biofilms in the system to ensure that any biocide tolerance of the system bacteria is reflected in the laboratory tests. In general, the results are borne out by the site audits of the control regime against sessile bacteria in the system.In an industrial system, biocide resistant populations develop from time to time. In practice it was found that even a small change to the biocide formulation could improve biocontrol, with concomitant reduction in corrosion rates and maintenance costs.     

Assessment of resistance towards biocides following the attachment of micro-organisms to, and growth on, surfaces

AIMS: To develop a rapid method for the assessment of biocidal activity directed towards intact biofilms. METHODS AND RESULTS: Escherichia coli and Staphylococcus epidermidis were cultured for up to 48 h within 96-well microtitre plates. The planktonic phase was removed and the wells rinsed. Residual biofilms were exposed to various concentrations of chloroxylenol, peracetic acid, polyhexamethylene biguanide (PHMB), cetrimide or phenoxyethanol for 1 h. At 15-min intervals, biocide was removed, and the wells washed in neutraliser and filled with volumes of fresh medium. Re-growth of the cultures was monitored during incubation at 35 degrees C in the plate reader. Times taken for the treated wells to re-grow to fixed endpoints were determined and related to numbers of surviving cells. Time--survival curves were constructed and the survival of the attached bacteria, following exposure to the agents for 30 min, interpolated for each biocide concentration. Log--log plots of these survival data and biocide concentration were constructed, and linear regression analysis performed in order to (i) calculate concentration exponents and (ii) compare the effectiveness of the biocides between variously aged biofilm and planktonic cells. From such analyses iso-effective concentrations of biocide (95% kill in 30 min) were calculated and expressed as planktonic : biofilm indices (PBI). CONCLUSION: PBI varied between 1.02 and 0.02, were relatively unaffected by age of the biofilms but differed significantly between organism and biocide. Notably those compounds with the higher activity against planktonic bacteria (PHMB and peracetic acid) were most prone to a biofilm effect but remained the most effective of the agents selected. SIGNIFICANCE AND IMPACT OF THE STUDY: The endpoint method proved robust, enabled the bactericidal effects of the biocides to be assessed against in-situ biofilms, and was suitable for routine screening applications.     

A green triple biocide cocktail consisting of a biocide, EDDS and methanol for the mitigation of planktonic and sessile sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) cause souring and their biofilms are often the culprit in Microbiologically Influenced Corrosion (MIC). The two most common green biocides for SRB treatment are tetrakis-hydroxymethylphosphonium sulfate (THPS) and glutaraldehyde. It is unlikely that there will be another equally effective green biocide in the market any time soon. This means more effective biocide treatment probably will rely on biocide cocktails. In this work a triple biocide cocktail consisting of glutaraldehyde or THPS, ethylenediaminedisuccinate (EDDS) and methanol was used to treat planktonic SRB and to remove established SRB biofilms. Desulfovibrio vulgaris (ATCC 7757), a corrosive SRB was used as an example in the tests. Laboratory results indicated that with the addition of 10–15% (v/v) methanol to the glutaraldehyde and EDDS double combination, mitigation of planktonic SRB growth in ATCC 1249 medium and a diluted medium turned from inhibition to a kill effect while the chelator dosage was cut from 2,000 to 1,000 ppm. Biofilm removal was achieved when 50 ppm glutaraldehyde combined with 15% methanol and 1,000 ppm EDDS was used. THPS showed similar effects when it was used to replace glutaraldehyde in the triple biocide cocktail to treat planktonic SRB.     

The use of biocides to control sulphate‐reducing bacteria in biofilms on mild steel surfaces

Three different types of biocides, viz. formaldehyde (FM), glutaraldehyde (GA) and isothiozolone (ITZ) were used to control planktonic and sessile populations of two marine isolates of sulphate‐reducing bacteria (SRB). The influence of these biocides on the initial attachment of cells to mild steel surfaces, on subsequent biofilm formation and on the activity of hydrogenase enzymes within developed biofilms was evaluated. In the presence of biocides the rate and degree of colonization of mild steel by SRB depended on incubation time, bacterial isolate and the type of biocide used. Although SRB differed in their susceptibility to biocides, for all isolates the biofilm population was more resistant to the treatment than the planktonic population. GA showed highest efficiency in controlling planktonic and sessile SRB compared with the other two biocides. The activity of the enzyme hydrogenase measured in SRB biofilms varied between isolates and with the biocide treatment. No correlation was found between the number of sessile cells and hydrogenase activity.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

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.     

Influence of a triazine derivative-based biocide on microbial biofilms of cutting fluids in contact with different substrates

Although biofilms are often associated with hospital infection problems owing to their high resistance to antimicrobial agents, in recent years biofilms have also been studied in the industrial sector, mainly because they are a major cause of contamination outbreaks in facilities and products. The aim of this study was to investigate whether different materials commonly found in the metalworking industries have different biofilm formation characteristics when in contact with contaminated cutting fluid as well as to establish an optimal concentration of a triazine-based antimicrobial agent to protect the oil/water emulsion and also to delay or interrupt the development of biofilms. Biofilms grown on the surface of carbon steel, stainless steel, aluminum, polyvinyl chloride, and glass were analyzed in terms of cell growth and susceptibility to the tested biocide. The results showed that the type of material used had little influence on cell adhesion or on the microbicide concentration required to control and eradicate microorganisms suspended in the emulsion and in the biofilms.     

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.     

Efficacy of Colloidal Silver-Hydrogen Peroxide and 2-Bromo-2-nitroporopane-1,3-diol Compounds Against Different Serogroups of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> Strains

Cooling towers are considered as amplifier and disseminator sources for Legionella spp. despite preventive treatments. Information which was obtained from biocidal tests could improve the effectiveness of treatments. Therefore, the choice of appropriate biocides and the applying of biocides in correct dosages and contact times are important. Various oxidizing and non-oxidizing biocides have been investigated in vitro for their effectiveness against legionellae. Colloidal silver-hydrogen peroxide (CSHP) and 2-bromo-2-nitroporopane-1,3-diol (BNPD) biocides were selected as an example for oxidizing and non-oxidizing agents, respectively, in view of bactericidal action against different serogroups of L. pneumophila strains [serogroup 1 (S1) and serogroup 2–14 (S2)] which are isolated different cooling towers in the vicinity of Istanbul, Turkey and reference strain. In the current study, oxidizing biocide was found more effective than non-oxidizing biocide in terms of contact times, log reductions and recommended dosages. At the recommended concentrations for cooling towers (100 ppm), while CSHP compound killed all strains in 3 h contact time, BNPD compound killed S2 and reference strain in the same contact time, S1 strain after 6 h contact time. The results of the present study showed that effective biocide applications can be achieved by pre-determining minimum inhibitory concentration (MIC) and minimum contact time of different biocides to kill target bacteria.     

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.     

Advances in the treatment of problematic industrial biofilms

In nature, microorganisms tend to form biofilms that consist of extracellular polymeric substances with embedded sessile cells. Biofilms, especially mixed-culture synergistic biofilm consortia, are notoriously difficult to treat. They employ various defense mechanisms against attacks from antimicrobial agents. Problematic industrial biofilms cause biofouling as well as biocorrosion, also known as microbiologically influenced corrosion. Biocides are often used to treat biofilms together with scrubbing or pigging. Unfortunately, chemical treatments suppress vulnerable microbial species while allowing resistant species to take over. Repeated treatment cycles are typically needed in biofilm mitigation. This leads to biocide dosage escalation, causing environmental problems, higher costs and sometimes operational problems such as scale formation. New treatment methods are being developed such as enhanced biocide treatment and bacteriophage treatment. Special materials such as antibacterial stainless steels are also being created to combat biofilms. This review discussed some of the advances made in the fight against problematic industrial 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.     

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step

Background

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.

Methodology/Principal Findings

Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.

Conclusions/Significance

These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.