Factors Affecting the Antimicrobial Activity of Disinfectants

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Chapter: Pharmaceutical Microbiology : Laboratory Evaluation Of Antimicrobial Agents

The activity of antimicrobial agents on a given organism or population of organisms will depend on a number of factors which must be reflected in the tests used to define their efficacy.



The activity of antimicrobial agents on a given organism or population of organisms will depend on a number of factors which must be reflected in the tests used to define their efficacy. For example, the activity of a given antimicrobial agent will be affected by nature of the agent, the nature of the challenge organism, the mode of growth of the challenge organism, concentration of agent, size of the challenge population and duration of exposure environmental/physical conditions (temperature, pH, presence of extraneous organic matter) are also important considerations in modelling the activity of biocidal agents. Laboratory tests for the evaluation of biocidal activity must be carefully designed to take into account these factors which may significantly influence the rate of kill within the microbial challenge population.


The work of Krönig and Paul in the late 1890s, demonstrated that the rate of chemical disinfection was related to both concentration of the chemical agent and the temperature of the system, and that bacteria exposed to a cidal agent do not die simultaneously but in an orderly sequence. This led to various attempts at applying the kinetics of pure chemical reactions (the mechanistic hypothesis of disinfection) to microbe/disinfectant inter-actions. However, since the inactivation kinetics depend on a large number of defined and undefined variables, such models are often too complicated for routine use. Despite this, the Chick-Watson model (equation 1), based on first-order reaction kinetics, remains the basic rate law for the examination of disinfection kinetics:


where N is the number of surviving microbes after time t and k0 is the disinfection rate constant. The Chick-Watson model may be further refined to account for biocide concentration (equation 2):



where k1 is the concentration-independent rate constant, B is the biocide concentration and n is the dilution coefficient. The Chick-Watson model predicts that the number of survivors falls exponentially at a rate governed by the rate constant and the concentration of disinfect-ant. A general assumption is that the concentration of biocide remains constant throughout the experiment; however, there are a number of situations when this appears not to be the case (sequestering) and may result in observed departures from linear reaction kinetics. The factors influencing the antimicrobial activity of disinfectant agents are discussed below.


a)   Innate (Natural) Resistance Of Microorganisms


The susceptibility of microorganisms to chemical disinfectants and biocides exhibits tremendous variation across various classes and species. Bacterial endospores and the mycobacteria (e.g. Mycobacterium tuberculosis) possess the most innate resistance, while many vegetative bacteria and some viruses appear highly susceptible. In addition, microorganisms adhering to surfaces as biofilms or present within other cells (e.g. legionellae within amoebae), may reveal a marked increase in resistance to disinfectants and biocides (Figure 18.3). Therefore, when evaluating new disinfectants, a suitable range of microorganisms and environmental conditions must be included in tests. The European suspension test (EN 12054) for hospital-related studies and the European/British Standard suspension test (EN 1276) for studies relating to food, industrial, institutional and domestic areas, both include Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae as the challenge organisms to be used in the test. For specific applications, additional strains may be chosen from Salmonella enterica serovar Typhimurium, Lactobacillus brevis and Enterobacter cloacae.



b)   Microbial Density


Many disinfectants require adsorption to the microbial cell surface prior to killing, therefore dense cell populations or sessile populations may sequester all the available disinfectant before all cells are affected, thus shielding a proportion of the population from the toxic effects of the chemical agent. Therefore, from a practical point of view, the larger the number of microorganisms present, the longer it takes a disinfectant to complete killing of all cells. For instance, using identical test conditions, it has been shown that 10 spores of the anthrax bacillus (Bacillus anthracis) were destroyed in 30 minutes, while it took 3 hours to kill 100 000 (105) spores. The implications of pre-disinfection washing and cleaning of objects (which removes most of the microorganisms) becomes obvious. However, when evaluating disinfectants in the laboratory, it must be remembered that unlike sterilization, kill curves with disinfectants may not be linear and the rate of killing may decrease at lower cell numbers (Figure 18.3). Hence a 3-log killing may be more rapidly achieved with 108 than 104 cells. Johnston et al. (2000) demonstrated that even small variations in the initial inoculum size (Staph. η= aureus) had a dramatic effect on log reductions over time, using a constant concentration of sodium dodecyl sulphate (SDS). The authors argue that the presence of microbes quenches the action of the biocide (self quenching), since cell and membrane components of lysed bacteria (e.g. emulsifiers such as triacylglycerols and phosphatidyl ethanolamine), are similar in action to emulsifiers (such as Tween and lecithin) used in standard biocide quenching/neutralizing agents employed in dis-infectant tests. However, this may not hold true across all biocides where similar inoculum size dependency of disinfection is observed (see Russell et al., 1997). Initial bioburden/cell numbers must, therefore, be standardized and accurately quantified in disinfectant efficacy (suspension) tests and agreement reached on the degree of killing required over a stipulated time interval (see Table 18.1).


c)      Disinfectant Concentration And Exposure Time


The effects of concentration or dilution of the active ingredient on the activity of a disinfectant are of major importance. With the exception of iodophors, the more concentrated a disinfectant, the greater its efficacy, and the shorter the time of exposure required to destroy the population of microorganisms, i.e. there is an exponential relationship between potency and concentration. Therefore, a graph plotting the log10 of the death time (i.e. the time required to kill a standard inoculum) against the log10 of the concentration is typically a straight line, the slope of which is the concentration exponent (η). Expressed as an equation:



Thus η can be obtained from experimental data either graphically or by substitution in equation (3) (see Table 18.2).


It is important to note that dilution does not affect the cidal attributes of all disinfectants in a similar manner. For example, mercuric chloride with a concentration exponent of 1 will be reduced by the power of 1 on dilution, and a threefold dilution means the disinfectant activity will be reduced by the value 31, i.e. to a third of its original activity. Phenol, however, has a concentration exponent of 6, so a threefold dilution in this case will mean a decrease in activity of 36 or 729 times less active than the original concentration. Thus, the likely dilution experienced by the disinfectant agent in use must be given due consideration when selecting an appropriate biocidal agent for a given application.


d)   Physical And Chemical Factors


Known and proven influences include temperature, pH and mineral content of water (‘hardness’).


i)   Temperature


As with most chemical/biochemical reactions, the cidal activity of most disinfectants increases with increase in temperature, since temperature is a measure of the kinetic energy within a reaction system. Increasing the kinetic energy of a reaction system increases the rate of reaction by increasing the number of collisions between reactants per unit time. This process is observed up to an optimum temperature, beyond which reaction rates fall again, due to thermal denaturation of some component(s) of the reaction. As the temperature is increased in arithmetical progression the rate (velocity) of disinfection increases in geometrical progression. Results may be expressed quantitatively by means of a temperature coefficient, either the temperature coefficient per degree rise in temperature (θ), or the coefficient per 10°C rise (the Q10 value) (Hugo & Russell, 1998). As shown by Koch, working with phenol and anthrax (B. anthracis) spores over 120 years ago, raising the temperature of phenol from 20°C to 30°C increased the killing activity by a factor of 4 (the Q10 value).


The value of θ may be calculated from the equation:


where t1 is the extinction time at T1°C, and t2 the extinction at T2°C (i.e. T1 + 1°C).


Q10 values may be calculated easily by determining the extinction time at two temperatures differing by exactly 10°C. Then:



It is also possible to plot the rate of kill against the temperature.


While the value for Q10 of chemical and enzyme-catalysed reactions lies in a narrow range (between 2 and 3), values for disinfectants vary widely, e.g. 4 for phenol, 45 for ethanol, and almost 300 for ethylene glycol monoethyl ether. Clearly, relating chemical reaction kinetics to disinfection processes is potentially dangerous. Most laboratory tests involving disinfectant-like chemicals are now standardized to 20°C, i.e. around ambient room temperatures.


ii)  pH


Effects of pH on antimicrobial activity can be complex. As well as directly influencing the survival and rate of growth of the microorganism under test, changes in pH may affect the potency of the agent and its ability to interact with cell surface sites. In a many cases (where the biocidal agent is an acid or a base), the ionization state (or degree of ionization) will depend on the pH. As is the case with some antimicrobials (e.g. phenols, acetic acid, benzoic acid), the non-ionized molecule is the active state (capable of crossing the cell membrane/partitioning) and alkaline pHs which favour the formation of ions of such compounds will decrease the activity. For these biocidal agents a knowledge of the molecule’s p Ka is important in predicting the pH range over which activity can be observed, since in situations where the pH of the system equals the p Ka of the biocide molecule, ionized and unionized species are in equilibrium. Others, such as glutaraldehyde and quaternary ammonium compounds (QACs), reveal increased cidal activity as the pH rises and are best used under alkaline conditions, possibly due to enhanced interaction with amino groups on microbial biomolecules. The pH also influences the properties of the bacterial cell surface, by increasing the proportion of anionic groups and hence its interaction with cidal molecules. Since the activity of many disinfectants requires adherence to cell surfaces; increasing the external pH renders cell surfaces more negatively charged and enhances the binding of cationic compounds such as chlorhexidine and QACs.


iii)  Divalent cations


The presence of divalent cations (e.g. Mg2+, Ca2+), for example in hard water, has been shown to exert an antagonistic effect on certain biocides while having an additive effect on the cidal activity of others. Metal ions such as Mg 2+ and Ca2+ may interact with the disinfectant itself to form insoluble precipitates and also interact with the microbial cell surface and block disinfectant adsorption sites necessary for activity. Biguanides, such as chlorhexidine, are inactivated by hard water. Hard water should always be employed for laboratory disinfectant and antiseptic evaluations to reflect this, with recommended formulae employing various concentrations of MgCl2 and CaCl2 solutions, available from the World Health Organization and the British Standard (BS EN 1276). On the other hand, cationic compounds may disrupt the outer membrane of Gram-negative bacteria and facilitate their own entry.


e)     Presence Of Extraneous Organic Material


The presence of extraneous organic material such as blood, serum, pus, faeces or soil is known to affect the cidal activity of many antimicrobial agents. Therefore, it is necessary to determine the likely interaction between organic matter and the disinfectant by including this parameter in laboratory evaluations of their activity. In order to simulate ‘clean’ conditions (i.e. conditions of minimal organic contaminant), disinfectants are tested in hard water containing 0.3 g/L bovine albumin, with the albumin being used to mimic ‘dirty’ conditions. This standardized method replaces earlier approaches, some of which employed dried human faeces or yeast to mimic the effects of blood, pus or faeces on disinfectant activity. Disinfectants whose activities are particularly attenuated in the presence of organic contaminant include the halogen disinfectants (e.g. sodium hypochlorite) where the disinfectant reacts with the organic matter to form inactive complexes, biguanides, phenolic compounds and QACs. The aldehydes (formaldehyde and glutaraldehyde) are largely unaffected by the presence of extraneous organic contaminants. Organic material may also interfere with cidal activity by adherence to the microbial cell surface and blockade of adsorption sites necessary for disinfectant activity. For practical purposes and to mirror potential in-use situations, disinfectants should be evaluated under both clean and dirty conditions. Alternatives to albumin have also been suggested, for example sheep blood or mucin.


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