One explanation for the variability of the bacterial suspension test

Disinfection kinetic studies of sodium dodecyl sulphate, benzalkonium chloride and sodium hypochlorite against Staphylococcus aureus revealed that when a higher inoculum level of Staph. aureus than normal was used (approximately 1 log higher), the efficacy of disinfection was severely attenuated. Kinetic analysis using the Hom model for experiments carried out on tests using 3 x 108 organisms ml-1 were unable to account for the large increase in disinfection power observed when smaller inoculum levels were used. Since the inoculum was the same in every way except for the numbers used, the large variations in the log reduction/time curves could not be explained by a variation in the resistance of the population to the biocide, as identical log reduction-time curves should have resulted. The level of disinfection achieved for a given concentration of biocide was found to be approximately linearly related to the cell number ml-1 of test solution and not to the log number. The variation observed is believed to occur due to intrinsic self-quenching of the biocide by the microbes during the course of the disinfection test. As the level of free biocide decreases, the rate of reaction decreases, giving the tails of the log reduction/time curves. Such intrinsic self-quenching could explain the large variations known to occur in the legally required disinfection suspension tests.     

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.     

Disinfectant testing: use of the Bioscreen Microbiological Growth Analyser for laboratory biocide screening

A new method is described for screening potential biocides based on the traditional suspension test using the Bioscreen optical plate reader. This new method is rapid, reproducible, quantitative and cost effective. Data obtained by this new method are not directly equivalent to the log reduction normally quoted, but give a measurement of the total effect of the biocide on the microbe population, measuring the effect of injury as well as death (non-viability). The method allows for the routine examination of disinfection kinetics, the study of which leads to greater scientific insight into disinfection than that achieved by the standard 5 min, one-point, disinfection tests currently employed.     

Adaptive responses to antimicrobial agents in biofilms

Bacterial biofilms demonstrate adaptive resistance in response to antimicrobial stress more effectively than corresponding planktonic populations. We propose here that, in biofilms, reaction-diffusion limited penetration may result in only low levels of antimicrobial exposure to deeper regions of the biofilm. Sheltered cells are then able to enter an adapted resistant state if the local time scale for adaptation is faster than that for disinfection. This mechanism is not available to a planktonic population. A mathematical model is presented to illustrate. Results indicate that, for a sufficiently thick biofilm, cells in the biofilm implement adaptive responses more effectively than do freely suspended cells. Effective disinfection requires applied biocide concentration that increases quadratically or exponentially with biofilm thickness.     

Biocide treatment of biofilms

Biofilms are ubiquitous in nature and microorganisms often exist as members of complex consortia, rather than as pure cultures. Their localised metabolic activity can create diffusion gradients of nutrients, fermentation byproducts and possible associated corrosion products within the biofilms; together with cell lysis, these cause a mosaic of microenvironments which may be totally different to the bathing phase. Such habitats pose a major, and often ignored, constraint on the interpretation of results obtained from laboratory disinfection models which can be physically, environmentally and physiologically inappropriate. For example, the most commonly used model for inactivation of microorganisms by biocides utilises the so-called ‘Chick- Watson law’: this implies that biocide concentration and contact time, the (C × T) factor, are the two key variables determining biocide efficacy. However, applications of the ‘law’ have assumed complete and uniform mixing of microorganisms and biocide, ignoring that diffusion might be rate limiting and that biocide concentration might decrease with time. Recent results suggest that many of the viable bacteria in chlorinated potable water are attached to surfaces and under these circumstances coliforms have withstood at least 12 ppm free residual chlorine. The use and efficacy of alternative biocides such as monochloramine against aquatic biofilms is discussed.     

Evidence of bacterial adaptation to monochloramine in Pseudomonas aeruginosa biofilms and evaluation of biocide action model

A mathematical model of biocide action against microbial biofilm was tested experimentally by measuring the response of Pseudomonas aeruginosa biofilm to various doses of monochloramine. Pure culture biofilm was developed in continuous flow annular reactors for 7 days, then treated with a 2-, 4-, or 8-h dose of 2 or 4 mg L(-1) monochloramine. Some experiments investigated repeated treatment. Disinfection and regrowth of the biofilm were observed by sampling the biofilm for viable and total cell areal densities for up to 100 h following the biocide treatment. A phenomenological mathematical model was fitted to experimental data sets and captured overall trends, but it could not simulate certain experimentally observed features. The model did simulate rapid disinfection followed by steady regrowth. It correctly predicted a much greater decrease in viable than in total cell densities and also correctly captured the shapes of these trajectories. Discrepancies between the model and data included the following: the model predicted faster regrowth than was experimentally observed, the model predicted that a second dose would be more effective than the first dose but the opposite was observed in the experiments, and parameters estimated by fitting one dose concentration could not be used to predict the results of a different dose concentration or a second dose. Discrepancies between model and the experiment were hypothesized to be due to an adaptive stress response by the bacteria, a process not included in the model. A practical implication of this work is that it is more effective to deliver monochloramine in a short concentrated dose as opposed to a longer dose of lower concentration. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 201-209, 1997.     

The effect of interfering substances on the disinfection process: a mathematical model

AIMS: To gain a greater understanding of the effect of interfering substances on the efficacy of disinfection. METHODS AND RESULTS: Current kinetic disinfection models were augmented by a term designed to quantify the deleterious effect of soils such as milk on the disinfection process of suspended organisms. The model was based on the assumption that inactivation by added soil occurred at a much faster rate than microbial inactivation. The new model, the fat-soil model, was also able to quantify the effect of changing the initial inoculum size (1 x 10(7)-5 x 10(7) ml(-1) of Staphylococcus aureus) on the outcome of the suspension tests. Addition of catalase to the disinfection of Escherichia coli by hydrogen peroxide, resulted in changes to the shape of the log survivor/time plots. These changes were modelled on the basis of changing biocide concentration commensurate with microbial inactivation. CONCLUSIONS: The reduction in efficacy of a disinfectant in the presence of an interfering substance can be quantified through the use of adaptations to current disinfection models. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the effect of soil on disinfection efficacy allows us to understand the limitations of disinfectants and disinfection procedures. It also gives us a mechanism with which to investigate the soil tolerance of new biocides and formulations.     

Efficacy of three prevention strategies against legionella in cooling water systems

The efficacy of three different prevention strategies on legionella in cooling systems was studied. The strategies were as follows: (1) water temperature was lowered; (2) water quality was improved; or (3) the system was disinfected with polyhexamethylene biguanidechloride (PHMB) biocide or with 2-bromo-2-nitropropane-1,3-diol (BNPD) biocide. Lowering of water temperature was the most effective method to reduce the concentration of legionella in cooling systems. Improving of water quality resulted in a transitory disinfection effect. The additions of PHMB or BNPD decreased the concentrations of both legionella and heterotrophic bacteria in cooling water. The effect of biocides, however, lasted at the most only a few months. If possible, lowering water temperature and improving the water quality should be the primary practices for controlling bacterial growth in cooling systems. Regular biocide treatments should be incorporated into the maintenance procedures if technical improvements cannot be done or if their efficiency is too low.     

Mechanisms of action of disinfectants

Disinfectants and biocides are a chemically diverse group of agents which are generally considered to exhibit poor selective toxicity. This should not be mistaken for poor target specificity, however, and much is now known concerning the damaging interactions which may arise between bacterial cell and disinfectant agent. Critical governing features of these interactions are the physicochemical characteristics of the chemical agent, cell morphology, and the physiological status of the microorganism. Antibacterial events include membrane disruption, macromolecule dysfunction, and metabolic inhibition; the consequential effect is determined by the relative contribution(s) of the target(s) to microbial cell survival and the possible initiation of self-destructive processes. Disinfection kinetics offer a measure to differentiate between physiochemical and chemical interactions.Increasingly demanding disinfectant applications require more sophisticated use of biocidal systems. Approaches include: agents in combination, whereby a knowledge of mechanism of action assists in designing optimal mixtures; intracellular biocide delivery, using cellular transport processes to overcome cellular barriers; and targeted donation of biocide from delivery systems, requiring an understanding of target reactivity.A knowledge of disinfection mechanisms provides a basis from which novel chemistries and synergistic combinations may be developed.     

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.     

An investigation into the differences between the Bioscreen and the traditional plate count disinfectant test methods

Investigations of biocide efficacy by automated methods involving optical density measurements, e.g. using the recently published 'Bioscreen' method, are complicated by the fact that a poor correlation often exists between the log reductions obtained using the automated method vs those obtained by the traditional plate count methods. It was hypothesized that the differences observed between the two methods were due to the level of cell injury, which was masked by the optical density methods but which was recognized by the plate counts. Comparisons of log reductions following a disinfection test always showed the Bioscreen method to be overestimating the log reductions with respect to the plate counts. A correlation between colony size on the plates and the 'Bioscreen' results for a fixed disinfectant concentration and contact time was found using Global Imaging software. The results obtained also suggested that the observed colony area was dependent on the amount of injury incurred by a microbe during the disinfection process. A mathematical model of injury was developed which predicted the observed differences between the Bioscreen and the traditional plate method. The model further suggested a possible reason for biocidal lags.     

Significance of biocide usage and antimicrobial resistance in domiciliary environments

Recent events have raised awareness of the need for effective hygiene in the home. Not least is the requirement to reduce antibiotic resistance by reducing the need for antibiotic prescribing. Current evidence suggests that improved hygiene in the domestic setting could have a significant impact. Recently, it has been suggested that widespread biocide usage, particularly in consumer products, may be a contributory factor in antibiotic resistance. In developing home hygiene policies, however, it is important that biocide use as an integral part of good hygiene practice is not discouraged in situations where there is real benefit. Although laboratory data indicate possible links, it is necessary to assess whether and to what extent biocide exposure could contribute to antibiotic resistance in clinical practice. The extent to which reduced susceptibility to biocides resulting from biocide exposure could compromise their 'in-use' effectiveness must also be considered. Equally, it is important that changes in susceptibility induced by biocide exposure are assessed relative to those induced by antibiotic exposure or the phenotypic changes induced by 'normal' environmental 'stresses'. It is proposed that to be effective, home hygiene policy should be based on the concept of risk assessment and risk prevention. Using this approach, critical risk situations are identified and appropriate hygiene procedures applied to reduce risks. This may involve either soap and water cleaning, or cleaning combined with a disinfection process. A 'targeted' hygiene approach not only provides the most effective means of preventing infectious disease, it also offers a means of addressing concerns about 'too much hygiene' and 'too many antibacterials' amongst a public who have lost confidence regarding appropriate hygiene for their home environment.     

Broad spectrum management of plant diseases by phylloplane microfungal metabolites

Plant surfaces are colonised by an array of microbes. These microbes have been implicated in protecting plants against invading pathogens. Studies have demonstrated that metabolites of phylloplane microfungi can induce defence responses in their host plants. This investigation relates to formulation of a biocide from metabolites of Aspergillus niger, Trichoderma viride, Alternaria alternata and Chaetomium globossum, which are common phylloplane colonisers of tropical plants. The biocide was found to reduce disease incidence through induction of alterations in defence physiology of crop plants.     

Disinfection in Food Processing – Efficacy Testing of Disinfectants

The key to effective cleaning and disinfection of food plants is the understanding of the type of the soil to be removed from the surfaces. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water with application of detergents and disinfectants. Disinfection is required in food plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The efficacy of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy a method for culturing laboratory model biofilms has developed. The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing can also be used for screening the activity of various disinfectants on biofilm cells. Both biofilm carrier tests showed clearly that the biofilm protects the microbes against the disinfectants. The chemical cleanliness is also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the microbial load using culturing techniques. These results can give an incorrect picture of the total cleanliness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent bacteria light inhibition method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection operations.     

Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae

Free-living amoebae (FLA) are found in most soil and aquatic environments. They claim our attention not only because they are intrinsically interesting microbes about which little is known but also because of the capacity for certain species to cause severe and often fatal disease in man. Naegleria fowleri produces acute meningoencephalitis, whilst Acanthamoeba spp. cause chronic encephalitis in immunocompromised individuals and also a destructive ocular infection associated with contact lens wear. FLA can also support the intracellular growth of Legionella pneumophila, the bacterium causing Legionnaires' disease in man. The ability to regulate the presence of pathogenic and nonpathogenic FLA in the environment throufh the use of chemical disinfection is therefore relevant in the prevention of human disease. In this paper the activity of water biocide chemicals and contact lens disinfectants against pathogenic FLA is presented.     

Apparent zero-order kinetics of phenol biodegradation by substrate-inhibited microbes at low substrate concentrations

The reaction kinetics for phenol biodegradation at low substrate concentrations can be estimated based on the analysis of changes in the dissolved oxygen concentration in the bulk liquid during biodegradation. The measured oxygen concentration changes with an interesting behavior as biodegradation proceeds. The oxygen concentration in the bulk liquid decreases rapidly in the early stages of degradation and subsequently decreases linearly and then rapidly recovers to the initial saturated level. Taking into account the oxygen transfer rate between gas and liquid phases and oxygen consumption rate by microbes, the change in the dissolved oxygen concentration can be simulated with an unsteady state mass balance equation and three kinetic models for the rate of phenol metabolism: a substrate-inhibited model; a zero-order model; and a combined model. In the combined model, it is assumed that, at phenol concentrations above 10 mg/L, the degradation rate is expressed by a substrate-inhibited model; whereas at concentrations below 10 mg/L the zero-order model is applied. It was found that the characteristics of the change in the dissolved oxygen concentration, especially the rapid increase at the end of degradation, can only be described by the combined kinetic model. This result suggests that conventional Haldane-type kinetics would be unsuitable for estimating the phenol consumption rate at low phenol concentrations, in particular, at concentrations less than 10 mg/L. (c) 1996 John Wiley & Sons, Inc.     

Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues

AIMS: The aims of this study were to evaluate the antimicrobial activity of chlorhexidine diacetate (CHX) and benzalkonium chloride (BZK) for strains of Pseudomonas aeruginosa exhibiting increased minimum inhibitory concentrations (MIC) for CHX, and to determine whether residues of chlorhexidine digluconate (CHG) and Hibiscrub (Hib, a formulation containing CHG) affect the susceptibility of P. aeruginosa to these biocides and a number of antibiotics. METHODS AND RESULTS: The bactericidal activity of CHX and BZK was evaluated for strains of P. aeruginosa exhibiting increased MIC for CHX with established suspension and surface disinfection tests. None of the strains of P. aeruginosa exhibiting raised MIC for CHX was less sensitive than the parent strain to CHX or BZK in either method. A test was designed to investigate the effects of dried CHG and Hib residues on P. aeruginosa cells. Exposure of P. aeruginosa to dried residues of CHG or Hib did not result in the organism becoming less sensitive to either biocide or a number of antibiotics. CONCLUSIONS: Pseudomonas aeruginosa strains with raised MIC to CHX were no less sensitive than the parent strain to CHX and BZK in bactericidal investigations. Exposure to dried residues of CHG and Hib did not render P. aeruginosa less sensitive to either of these agents or a number of antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: An increase in the MIC for a biocide in a micro-organism does not necessarily result in a failure of the biocide to effectively kill the organism. The residue that remains after the use of an antimicrobial agent can be at a far lower concentration than that initially applied and this study highlights the necessity for further investigations into the effect of residues, at low concentration, on bacterial populations and their role, if any, in the continued problem of antibiotic resistance.     

Rapid assay for the assessment of a potential of chemical biocides to microbial destructors of industrial materials

A colorimetric rapid assay for estimating the biocide potential of various chemicals towards metal biocorrosive and petroleum product degrading microbes was developed based on the reducing potential of live microbial cell. A water-soluble organic redox indicator, blue in the oxidized form and pink in the reduced form, was used as an indicator of the reducing potential of microbial cells. Once added to a suspension of vital microbial cells, it was reduced and changed in color. A good correlation between the results of this assay and viability control was obtained by employing surfactants and heavy metal ions.     

Rapid Assay for Assessment of the Potential of Chemical Biocides against Microbial Destructors of Industrial Materials

A colorimetric rapid assay for estimating the biocide potential of various chemicals towards metal- biocorrosive and petroleum-product-degrading microbes was developed based on the reducing potential of live microbial cells. A water-soluble organic redox indicator, blue in the oxidized form and pink in the reduced form, was used as an indicator of the reducing potential of microbial cells. Once added to a suspension of vital microbial cells, it was reduced and changed in color. A good correlation between the results of this assay and viability control was obtained by employing surfactants and heavy metal ions.