Laboratory Evaluation Of Antimicrobial Agents PDF

366 19 Laboratory Evaluation of Antimicrobial Agents 19.1 ­Introduction mode of growth are phenotypically distinct from their planktonic counterparts and frequently exhibit...

366 19 Laboratory Evaluation of Antimicrobial Agents 19.1 ­Introduction mode of growth are phenotypically distinct from their planktonic counterparts and frequently exhibit significantly Laboratory evaluation of antimicrobial agents remains a elevated phenotypic tolerance to antimicrobial challenge cornerstone of clinical microbiology and antimicrobial/ (see Chapter 8). This has implications not only for the envi- microbicide discovery and development. The establishment ronmental control of microorganisms but importantly in the of robust and reproducible assays for determining microbial selection of appropriate concentrations of antibiotic or susceptibility to antimicrobial agents is of fundamental microbicide necessary to eradicate them. As such, biofilms importance in the appropriate selection of therapeutic may constitute a reservoir of infectious microorganisms agents and microbicides for use in infection prevention and which may persist following antimicrobial challenge, espe- control, disinfection, preservation and antifouling applica- cially where antimicrobial selection is based on standard tions. Such laboratory assays form the basis for high-­ laboratory susceptibility tests on planktonic cultures of throughput screening of compounds or biological extracts in microorganisms. Tests for evaluating candidate antimicro- the discovery, isolation and development of new antimicro- bial agents to be used in human and animal medicine as well bial drugs and microbicides. Antimicrobial screening assays as environmental microbicides remain significant labora- facilitate identification of antimicrobial agents from various tory considerations. sources and in lead antimicrobial compound optimisation. In the control of human and animal infection, laboratory 19.1.1 Definitions evaluation of candidate agents yields crucial information which can inform the choice of antimicrobial agent(s) where Key terms such as disinfection, preservation, antisepsis the causative organism is known or suspected. As the num- and sterilisation are defined in Chapters 18 and 21. A num- ber of microorganisms exhibiting resistance to conventional ber of other important terms used to describe the antimi- antimicrobial agents increases, laboratory evaluation of crobial activity of agents are also commonly used. A biocide antimicrobial susceptibility is increasingly important for the may be defined as a chemical or physical agent which kills selection of appropriate therapeutic agents. Evaluation of viable organisms, both pathogenic and nonpathogenic. the potential antimicrobial action and nature of the inhibi- This broad definition clearly includes microoganisms, but tory or lethal effects of established and novel therapeutic is not restricted to them. The term microbicide is therefore agents and microbicides are important considerations in the also used to refer specifically to an agent which kills micro- success of therapeutic interventions and infection/contami- organisms (germicide may also be used in this context, but nation control procedures. generally refers to pathogenic microorganisms). The terms Significant concerns that the extensive use of microbi- bactericidal, fungicidal, microbicidal and viricidal therefore cidal agents may be linked to the development of antimi- describe an agent with killing activity against a specific crobial resistance exist (see Chapter 20). Recent concerns class or classes of organism indicated by the prefix, whereas regarding significant global public health issues such as the the terms bacteriostatic and fungistatic refer to agents increasing threat of bioterrorism, the prevalence of which inhibit the growth of bacteria or fungi, respectively healthcare-­associated infections, avian influenza (H5N1), (Figure 19.1), but do not necessarily kill them. It should be swine flu (H1N1) and especially the COVID-­19 pandemic, noted, however, that some microorganisms that appear caused by the coronavirus SARS-­CoV-­2, have seen global non-­viable and non-­cultivable following antimicrobial demand for microbicides and novel biocidal technologies challenge may be revived by appropriate methods, and that increase dramatically. In addition, the emergence of new microorganisms incapable of multiplication may retain infectious agents (e.g., prions) and the increasing transmis- some metabolic/enzymatic activity. Bacteria also exhibit a sion rates of significant blood-­borne viruses (e.g., HIV, stress-­induced dormant phenotype, known as the persister hepatitis B and C), which may readily contaminate medical phenotype or persisters, whereby they neither grow nor are instruments or the environment, have focused attention on killed by exogenous stress such as starvation or antimicro- the need for effective and proven disinfecting and sterilis- bial challenge. This persister phenotype allows members of ing agents. a population of microbial cells (bacteria, archaea) to sur- Finally, increased appreciation of the role played by micro- vive highly elevated concentrations of antibiotic or antimi- bial biofilms in human and animal infectious diseases and crobial agent, and then to resuscitate after the exogenous their ubiquitous distribution in natural ecosystems have led stressor is removed and more favourable growth conditions to the development of novel approaches for the laboratory return. Recent research has shown that rather than being evaluation of antimicrobial susceptibility of microorganisms two distinct stress-­induced phenotypes, the viable but non-­ growing as surface-­adhered sessile populations. These stud- culturable (VBNC) and persister phenotypes actually ies have demonstrated that microorganisms in the biofilm describe the same dormant phenotype.  19.1 ­Introductio 367 microbial biofilm. For conventional antibiotics and ­microbicides, the MBEC value may be 1000-­fold higher than the MBC for the same planktonic microorganisms. Further studies have shown that often no correlation Normal growth exists between the MIC and the MBEC, indicating the Viable cells (log) potential limitations of therapeutic antibiotic selection based on determined MIC values. Analogous to the MIC, the minimum biofilm inhibitory concentration (MBIC) is Effect of a suggested as an additional biofilm susceptibility endpoint static agent parameter, and is defined as the lowest concentration of an antibiotic, or antimicrobial substance, at which there is Effect of a no ­time-­dependent increase in the number of biofilm cidal agent cells. However, unlike the MBEC value which is a ­well-­established biofilm susceptibility endpoint ­parameter, there is a lack of consistency in the literature regarding x Time (hours) MBIC determination protocols and definition, with an alternative definition of MBIC as the concentration of Figure 19.1 Effect on the subsequent microbial growth pattern antibiotic that displays biofilm inhibition of >90% based of inhibitory (static, Δ) or cidal (◻) agents added at time X (the normal microbial growth pattern is indicated by the ⚬ line). on CFU (colony-­forming units) or RFU (relative ­fluorescence units in a resazurin assay) determinations. The term tolerance implies the ability of some bacterial strains to survive (without using or expressing resistance In the laboratory evaluation of antibacterial agents, the mechanisms), but not grow, at levels of antimicrobial terms minimum inhibitory concentration (MIC) and mini- agent that should normally be cidal. This applies particu- mum bactericidal concentration (MBC) are most com- larly to systems employing the cell-­wall-­active β-­lactams monly used. The British Society for Antimicrobial and glycopeptides, and to Gram-­positive bacteria such as Chemotherapy (BSAC) guidelines for the determination streptococci. Normally, MIC and MBC levels in such tests of minimum inhibitory concentrations define the MIC should be similar (i.e., within one or two doubling dilu- as the lowest concentration of antimicrobial which tions); if the MIC/MBC ratio is 32 or greater, the term will inhibit the visible growth of a microorganism after tolerance is used. Tolerance may in some way be related overnight cultivation, and the MBC as the lowest concen- to the Eagle phenomenon (paradoxical effect), where tration of antimicrobial that will prevent the recovery and increasing concentrations of antimicrobial result in growth of a microorganism after subculture onto reduced killing rather than the increase in cidal activity antibiotic-­free media. Generally, MIC and MBC values are expected (see Figure 19.2). Tolerance to elevated antimi- recorded either in milligrams per litre or per millilitre crobial challenge concentrations is also a characteristic (mg l–1 or mg ml–1) or in micrograms per microlitre (μg μl–1). of microbial biofilm populations. Finally, the term With most cidal antimicrobials, the MIC and MBC are fre- ­resistance has several definitions within the literature; quently near or equal in value, although with essentially however, it generally refers to the ability of a microorgan- static agents (e.g., tetracycline), the lowest concentration ism to withstand the effects of a harmful chemical agent, required to kill the microorganism (i.e., the MBC) is invar- with the organism neither killed nor inhibited at concen- iably many times the MIC and often clinically unachieva- trations to which the majority of strains of that organism ble without toxicity to the human host. As with are susceptible. In the case of bacteria, resistance is microbicides, cidal terms can be applied to studies involv- defined as resistance to an antibiotic or antimicrobial ing not just bacteria but other microbes, for example, agent that was once capable of treating an ­infection when referring to cidal antifungal agents, the term mini- caused by that bacterial strain. Resistance mechanisms mum fungicidal concentration (MFC) is used. Recently, generally involve modification of the ­normal target of thanks to developments in the design of high-­throughput the antimicrobial agent either by mutation, enzymatic laboratory screens for biofilm susceptibility, the minimum changes, target substitution, antibiotic destruction or biofilm eradication concentration (MBEC) can be accu- alteration, antibiotic efflux mechanisms or restricted rately determined for organisms grown as single-­ or permeability to antibiotics. Antibiotic-­resistance mecha- mixed-­species biofilms. The MBEC is the minimum con- nisms are discussed fully in Chapter 13 and those for centration of an antimicrobial agent required to kill a microbicides in Chapter 20. 368 19 Laboratory Evaluation of Antimicrobial Agents 100 interfering substances [e.g., divalent cations] and exoge- nous organic matter [EOM]). The work of Krönig and Paul in the late 1890s demon- strated that the rate of chemical disinfection was related to 10 both concentration of the chemical agent and the tempera- ture of the system, and that bacteria exposed to a cidal agent do not die simultaneously but in an orderly sequence. Log survival (%) This led to various attempts at applying the kinetics of pure chemical reactions (the mechanistic hypothesis of disinfec- 1 tion) to microbe/disinfectant interactions. However, since the inactivation kinetics depend on a large number of defined and undefined variables, such models are often too 0.1 complicated for routine use. Despite this, the Chick– Watson model (Equation (19.1)), based on first-­order reac- tion kinetics, remains the basic rate law for the examination of disinfection kinetics: 0.01 dN k0 N (19.1) 0 1 10 100 dT Antimicrobial concentration (mg l–1) where N is the number of surviving microbes after time t Figure 19.2 Survival of Enterococcus faecalis exposed to a and k0 is the disinfection rate constant. The Chick–Watson fluoroquinolone for 4 hours at 37 °C. Three initial bacterial model may be further refined to account for microbicide concentrations were studied, 107 CFU ml–1 (◻); 106 CFU ml–1 (Δ) concentration (Equation (19.2)): and 105 CFU ml–1 (⚬). This clearly demonstrates a paradoxical effect (increasing antimicrobial concentrations past a critical level reveal decreased killing), and the effects of increased dN (19.2) k1C n N inoculum densities on subsequent killing. Source: Courtesy of dT Dr. Z. Hashmi. where k1 is the concentration-­independent rate constant, C is the microbicide concentration and n is the dilution 19.2 ­Factors Affecting the Antimicrobial ­coefficient. The Chick–Watson model predicts that the Activity of Disinfectants number of survivors falls exponentially at a rate governed by the rate constant and the concentration of disinfectant. The activity of antimicrobial agents against a given organ- A general assumption is that the concentration of ism or population of organisms will depend on a number of ­microbicide remains constant throughout the experiment; factors which must be reflected in the tests used to define ­however, there are a number of situations when this their efficacy. For example, the activity of a given antimicro- appears not to be the case (e.g., sequestering) and may bial agent will be affected by the nature of the agent, the result in observed departures from linear reaction kinetics. characteristics of the challenge organism, the mode of The factors influencing the antimicrobial activity of disin- growth of the challenge organism, concentration of the fectant agents are discussed below. agent, size of the challenge population and duration of exposure of that population to the active agent. Furthermore, 19.2.1 Innate (Natural) Resistance of Microorganisms environmental/physical conditions (temperature, pH, pres- ence of extraneous organic matter) are also important con- The susceptibility of microorganisms to chemical disinfect- siderations in modelling the activity of microbicidal agents. ants and microbicides exhibits tremendous variation across Laboratory tests for the evaluation of microbicidal activity various classes, species, phenotypes and morphologies. must be carefully designed to take into account these factors Bacterial endospores and the mycobacteria (e.g., which may influence significantly the rate of kill within the Mycobacterium tuberculosis) exhibit the highest innate microbial challenge population. In other words, disinfect- resistance, while many vegetative bacteria and some viruses ant efficacy tests must be designed to mimic likely in-­use appear highly susceptible (see Chapter 18). In addition, conditions given their intended applications (those param- microorganisms adhering to surfaces as biofilms or present eters encountered during routine use including surface within other cells (e.g., legionellae within ­amoebae) materials, challenge microorganisms, pH, presence of may show a marked increase in phenotypic resistance  19.2 ­Factors Affecting the Antimicrobial Activity of Disinfectant 369 100 Table 19.1 Methods of recording viable cells remaining after Control exposure of an initial population of 1,000,000 (106) CFU to a cidal agent. 10 Amoebae grown cells Viable count Log survival (%) remaining (CFU) Log survival (%) Log killing % killing 1 100,000 (105) 10 1-­log 90 4 10,000 (10 ) 1 2-­log 99 Broth grown cells 1000 (103) 0.1 3-­log 99.9 2.1 100 (10 ) 0.01 4-­log 99.99 10 (101) 0.001 5-­log 99.999.01 0 1 2 4 6 well as extraneous organic material) become obvious. Time (hours) However, when evaluating disinfectants in the laboratory, Figure 19.3 Survival of stationary phase broth cultures of it must be remembered that unlike sterilisation, kill curves Legionella pneumophila and amoebae-­grown L. pneumophila with disinfectants may not be linear and the rate of killing after exposure to 32 mg l–1 benzisothiazolone (Proxel) at 35 °C may decrease at lower cell numbers (Figure 19.3). Hence, a in Ringer’s solution; the control has no microbicide present. 3-­log killing may be more rapidly achieved with 108 than Source: Adapted from Barker et al. (1992). 104 cells. Johnston et al. (2000) demonstrated that even small variations in the initial inoculum size (S. aureus) had a dramatic effect on log reductions over time, using a con- (or tolerance) to disinfectants and microbicides stant concentration of sodium dodecyl sulphate (SDS). The (Figure 19.3). Therefore, when evaluating new disinfect- authors argue that the presence of microbes quenches the ants, a suitable range of microorganisms and environmen- action of the biocide (self-­quenching), since cell and mem- tal conditions must be included in tests to again mimic brane components of lysed bacteria (e.g., emulsifiers such in-­use conditions rather than optimal laboratory ­conditions, as triacylglycerols and phosphatidylethanolamine) are the results of which may not be reflective of microbicide similar in action to emulsifiers (such as Tween and leci- performance in practice. The European/British Standard thin) used in standard microbicide quenching/neutralising suspension test (BS EN 1276:2019) for studies relating to agents employed in disinfectant tests. However, this may food, industrial, institutional and ­domestic areas include not hold true across all microbicides where similar Pseudomonas aeruginosa, Escherichia coli, Staphylococcus ­inoculum size dependency of disinfection is observed aureus and Enterococcus hirae as the challenge organisms to (see Russell et al. 1997). Initial bioburden/cell numbers be used in the test. For specific applications, additional must, therefore, be standardised and accurately quantified strains may be chosen from Salmonella enterica serovar in disinfectant efficacy (suspension) tests and agreement Typhimurium, Lactobacillus brevis and Enterobacter reached on the degree of killing required over a stipulated cloacae. time interval (see Table 19.1). 19.2.2 Microbial Density 19.2.3 Disinfectant Concentration and Exposure Time Many disinfectants require adsorption to the microbial cell surface prior to killing; therefore, dense cell populations or The effects of concentration or dilution of the active sessile populations may sequester all, or a significant pro- ­ingredient on the activity of a disinfectant are of major portion, of the available disinfectant before all cells are importance. With the exception of iodophors, the more affected, thus shielding a proportion of the population concentrated a disinfectant, the greater its efficacy and the from the toxic effects of the chemical agent. Therefore, shorter the exposure time required to destroy the popula- from a practical point of view, the larger the number of tion of microorganisms, that is, there is a direct relation- microorganisms present, the longer it takes a disinfectant ship, frequently exponential, between potency and to bring about complete killing of the population of cells. concentration. Therefore, a graph plotting the log10 of the The implications of pre-­disinfection washing and cleaning death time (i.e., the time required to kill a standard of objects (which remove most of the microorganisms, as ­inoculum) against the log10 of the concentration is 370 19 Laboratory Evaluation of Antimicrobial Agents Table 19.2 Concentration exponents, η, for some disinfectant agent for a given application. It should also be remembered substances. that sequestration of the active biocide by organic matter, or an increased bioburden, may also constitute dilution Antimicrobial agent Concentration exponent events which need to be considered when evaluating microbicides under in-­use conditions. Hydrogen peroxide 0.5 Silver nitrate 0.9–1.0 Mercurials 0.03–3.0 19.2.4 Physical and Chemical Factors Iodine 0.9 Known and proven influences on microbicidal efficacy Crystal violet 0.9 include temperature, pH and mineral content of water Chlorhexidine 2 (‘hardness’). Formaldehyde 1 QACs 0.8–2.5 19.2.4.1 Temperature Acridines 0.7–1.9 As with most chemical/biochemical reactions, the cidal Formaldehyde donors 0.8–0.9 activity of most disinfectants increases with an increase in Bronopol 0.7 temperature, since temperature is a measure of the kinetic Polymeric biguanides 1.5–1.6 energy within a reaction system. Increasing the kinetic energy of a reaction system increases the rate of reaction by Parabens 2.5 increasing the number of collisions between reactants per Sorbic acid 2.6–3.2 unit time. This process is observed up to an optimum tem- Potassium laurate 2.3 perature, beyond which reaction rates fall again, due to Benzyl alcohol 2.6–4.6 thermal denaturation of some component(s) of the reac- Aliphatic alcohols 6.0–12.7 tion. As the temperature is increased in arithmetical Glycol monophenyl ethers 5.8–6.4 ­progression, the rate (velocity) of disinfection increases in Glycol monoalkyl ethers 6.4–15.9 geometrical progression. Results may be expressed quanti- Phenolic agents 4.0–9.9 tatively by means of a temperature coefficient, either the temperature coefficient per degree rise in temperature (θ), QAC, quaternary ammonium compound. or the coefficient per 10 °C rise (the Q10 value). As shown by Koch, working with phenol and anthrax (Bacillus anthracis) spores over 120 years ago, raising the tempera- typically a straight line, the slope of which is the concentra- ture of phenol from 20 to 30 °C increased the killing activ- tion exponent (η). This is expressed as an equation: ity by a factor of 4 (the Q10 value). log death time at concentration C2 The value of θ may be calculated from the equation: log death time at concentration C1 (19.3) T1 T2 t1 / t2 (19.4) logC1 C2 where t1 is the extinction time at temperature T1 °C, and t2 Thus, η can be obtained from experimental data either the extinction at T2 °C (i.e., T1 + 1 °C). graphically or by substitution in Equation (19.3) (see Q10 values may be calculated easily by determining the Table 19.2). extinction time at two temperatures differing by exactly It is important to note that dilution does not affect the 10 °C. Then: cidal attributes of all disinfectants in a similar manner. For example, mercuric chloride with a concentration exponent Time to kill at T Q10 (19.5) of 1 will be reduced by the power of 1 on dilution, and a Time to kill at T 10 threefold dilution means the disinfectant activity will be reduced by the value 31, that is, to a third of its original It is also possible to plot the rate of kill against the potency. Phenol, however, has a concentration exponent of temperature. 6, and so a threefold dilution in this case will mean a While the value of Q10 for chemical-­ and enzyme-­ decrease in activity of 36 or 729 times less active than the catalysed reactions lies in a narrow range (between 2 and original concentration. Thus, the likely dilution experi- 3), values for disinfectants vary widely, for example, 4 for enced by the disinfectant agent in use must be given due phenol, 45 for ethanol and almost 300 for ethylene glycol consideration when selecting an appropriate microbicidal monoethyl ether. Clearly, relating chemical reaction  19.3 ­Evaluation of Liquid Disinfectant 371 kinetics without qualification to disinfection processes is 19.2.5 Presence of Extraneous Organic Material potentially misleading. Most laboratory tests involving The presence of extraneous organic material such as disinfectant-­like chemicals are now standardised to 20 °C, blood, serum, pus, faeces or soil is known to affect the that is, around ambient room temperatures. cidal activity of many antimicrobial agents. Therefore, it is necessary to determine the likely interaction between 19.2.4.2 pH organic matter and disinfectants by including this param- Effects of pH on antimicrobial activity can be complex. As eter in laboratory evaluations of their activity. In order to well as directly influencing the survival and rate of growth simulate ‘clean’ conditions (i.e., conditions of minimal of the microorganism under test, changes in pH may affect organic contamination), disinfectants are tested in hard the potency of the agent and its ability to interact with cell water containing 0.3 g/l bovine serum albumin (BSA), surface sites. In many cases (for instance, where the micro- while 3.0 g/l BSA is used to mimic ‘dirty’ conditions. This bicidal agent is an acid or a base), the ionisation state (or standardised method replaces earlier approaches, some of degree of ionisation) will depend on the pH. As is the case which employed dried human faeces or yeast to mimic the with some antimicrobials (e.g., phenols, acetic acid, ben- effects of blood, pus or faeces on disinfectant activity. zoic acid), the non-­ionised molecule is the active state Disinfectants whose activities are particularly attenuated (capable of crossing the cell membrane/partitioning) and in the presence of organic contaminant include the halo- alkaline pHs which favour the formation of ions of such gen disinfectants, for example, sodium hypochlorite compounds will decrease the activity. For these microbi- (where the disinfectant reacts with the organic matter to cidal agents, a knowledge of the molecule’s pKa is impor- form inactive complexes), biguanides, phenolic com- tant in predicting the pH range over which activity can be pounds and QACs. The aldehydes (formaldehyde and glu- observed, since in situations where the pH of the system taraldehyde) are largely unaffected by the presence of equals the pKa of the biocide molecule, ionised and union- extraneous organic contaminants. Organic material may ised species are in equilibrium. Others, such as glutaralde- also interfere with cidal activity by coating the microbial hyde and quaternary ammonium compounds (QACs), cell surface blocking adsorption sites necessary for disin- reveal increased cidal activity as the pH rises and are best fectant activity. For practical purposes and to mirror used under alkaline conditions, possibly due to enhanced potential in-­use situations, disinfectants should be evalu- interaction with amino groups on microbial biomolecules. ated under both clean and dirty conditions. The pH also influences the properties of the bacterial cell surface by changing the proportion of anionic groups pre- sent and hence altering its interaction with cidal molecules. 19.3 ­Evaluation of Liquid Disinfectants Since the activity of many disinfectants first requires attachment to cell surfaces, increasing the external pH ren- 19.3.1 General ders those surfaces more negatively charged and thus enhances the binding of cationic compounds such as chlo- Phenol coefficient tests were developed in the early twen- rhexidine and QACs. tieth century when typhoid fever was a significant public health problem and phenolics were regularly used to dis- 19.2.4.3 Divalent Cations infect contaminated utensils and other inanimate objects. The presence of divalent cations (e.g., Mg2+, Ca2+), for In these tests, alternative disinfecting agents were com- example, in hard water, has been shown to exert an pared to a phenol standard; details of such tests can be antagonistic effect on certain microbicides while having found in earlier editions of this book. As non-­phenolic an additive effect on the cidal activity of others. Metal disinfectants became more widely available, however, ions such as Mg2+ and Ca2+ may interact with the disin- tests that more closely paralleled the conditions under fectant itself to form insoluble precipitates; they can which disinfectants were being used (e.g., blood spills) also interact with the microbial cell surface and block and which included a more diverse range of microbial disinfectant adsorption sites necessary for activity. types (e.g., viruses, bacteria, fungi, protozoa) were devel- Biguanides, such as chlorhexidine, are inactivated by oped. Evaluation of a disinfectant needs to be based on its hard water. Hard water should always be employed for ability to kill microbes, that is, its cidal activity, under laboratory disinfectant and antiseptic evaluations to environmental conditions mimicking as closely as possi- best reflect the in-­use situation; recommended formu- ble real-­life situations. As an essential component of each lae for such tests employing various concentrations of test is a final viability assay, removal or neutralisation of MgCl2 and CaCl2 solutions can be found in the British any residual disinfectant (to prevent ‘carry-­over’ toxicity) Standard BS EN 1276:2019. becomes a significant consideration. 372 19 Laboratory Evaluation of Antimicrobial Agents The development of methods to evaluate disinfectant has been suggested for suspension tests, some authorities activity in diverse environmental conditions and to deter- require a 6-­log killing in simulated use tests. With viruses, mine suitable in-­use concentrations/dilutions to be used a 4-­log killing tends to be an acceptable result, while with led to the development by Kelsey, Sykes and Maurer of the prions it has been recommended that a titre loss of 104 pri- so-­called capacity-­use dilution test which measured the ons should be regarded as an indication of appropriate dis- ability of a disinfectant at appropriate concentrations to infection provided that there has been adequate prior kill successive additions of a bacterial culture. Results were cleaning. With simulated use tests, cleaning followed by reported simply as pass or fail and not a numerical coeffi- appropriate disinfection should result in a prion titre loss cient. Tests employed disinfectants diluted in hard water of at least 107. (clean conditions) and in hard-­water-­containing organic material (yeast suspension to simulate dirty conditions), 19.3.2 Antibacterial Disinfectant Efficacy Tests with the final recovery broth containing 3% Tween 80 as a neutraliser. Such tests are applicable for use with a wide Various regulatory authorities in Europe (e.g., European variety of disinfectants (see Kelsey and Maurer 1974). Standard or Norm [EN]; British Standards [BS]; Germany Capacity tests mimic the practical situations of housekeep- [Deutsche Gesellschaft für Hygiene und Mikrobiologie, ing and instrument disinfection, where surfaces are DGHM]; France [Association Française de Normalisation, ­contaminated, exposed to disinfectant, recontaminated AFNOR]) and North America (e.g., Food and Drug and so forth. The British Standard (BS 6905:1987) method Administration [FDA]; EPA; ASTM International [for- for estimation of disinfectants used in dirty conditions in merly the American Society for Testing and Materials] and hospitals by a modification of the original Kelsey–Sykes AOAC) have been associated with attempts to produce test is one of the most commonly employed capacity tests some form of harmonisation of disinfectant tests. The in the UK and Europe. In the USA, effectiveness test data European Standard methods for disinfectant validation for submission to the US Environmental Protection Agency have been widely adopted, however, and serve as a useful (EPA) for disinfectant claim substantiation must be illustration of the general approach; they comprise three obtained by methods accepted by the Association of phases (also referred to as tiers). Phase 1 disinfectant effi- Official Analytical Chemists (AOAC), known collectively cacy tests are typically performed at the developmental as disinfectant efficacy tests (DETs). stage of a disinfectant formulation to determine whether The best information concerning the fate of microbes an active agent qualifies for basic disinfectant claims based exposed to a disinfectant, however, is obtained by counting on effectiveness against specified challenge microorgan- the number of viable cells remaining after exposure of a isms. Two basic Phase 1 tests are described in the EN stand- standard suspension of those cells to the disinfectant at ard documents EN 1040:2005 (bactericidal activity) and known concentration for a given time interval; these are EN1275:2005 (fungicidal/yeasticidal activity). These tests called suspension tests. Viable counting is a facile technique are conducted under laboratory conditions, with no addi- used in many branches of pure and applied microbiology tion of interfering substances, and as such may not be used (see Chapter 2). Assessment of the number of viable for efficacy claims, since the tests are not designed to mimic microbes remaining (survivors) after exposure allows the in-­use conditions. Phase 2 tests are divided further into killing or cidal activity of the disinfectant to be expressed in Steps 1 and 2 tests where Phase 2, Step 1 tests are quantita- a variety of ways, for example, percentage kill (e.g., tive suspension tests which simulate in-­use environmental 99.999%), as a log10 reduction in numbers (e.g., 5-­log kill- conditions using interfering substances such as BSA; here, ing), or by log10 survival expressed as a percentage. EN 13272:2012 and EN 13624:2013 evaluate bactericidal Examples of such outcomes are shown in Table 19.1. and fungicidal activities, respectively, and EN 14348 Unfortunately, standardisation of the methodology to be describes quantitative suspension tests for mycobacteri- employed in these efficacy tests has proven difficult to cidal activity. Phase 2, Step 2 tests are carrier tests intended obtain, as has consensus on what level of killing represents to simulate practical usage conditions, whereby a defined a satisfactory and/or acceptable result. It must be stressed, inoculum of the challenge organism is applied to non-­ however, that unlike tests involving chemotherapeutic porous surfaces (the carrier) and left to air-­dry, with the agents where the major aim is to establish antimicrobial disinfectant being subsequently applied for designated concentrations that inhibit growth (i.e., MICs), disinfect- contact times. Interfering substances are included in the ant tests require determinations of appropriate cidal levels. test to mimic environmental soiling. Examples of Phase 2, Levels of killing required over a given time interval tend to Step 2 standard European methods are EN 14561:2006, vary depending on the regulatory authority concerned. 14562:2006 and 14563 quantitative carrier tests for While a 5-­log killing of bacteria (starting with 106 CFU ml–1) ba­ctericidal, fungicidal/yeasticidal and mycobactericidal  19.3 ­Evaluation of Liquid Disinfectant 373 activities, respectively; these tests are applied for disinfect- initially decline in numbers in diluents devoid of ­additional ing medical instruments (the carrier material). disinfectant, results from tests incorporating disinfectant-­ treated cells can be compared with results from simultane- 19.3.2.1 Suspension Tests ous tests involving a non-­disinfectant-­containing system While varying to some degree in their methodology, most (untreated cells). The bactericidal effect BE can then be procedures employ a standard suspension of the microor- expressed as: ganism in hard water containing albumin (dirty condi- BE log N C log N D (19.6) tions) and appropriate dilutions of the disinfectant. Tests are carried out at a set temperature (usually around room where NC and ND represent the final number of CFU ml–1 temperature or 20 °C), and at a selected time interval sam- remaining in the control and disinfectant series, ples are removed and viable counts are performed follow- respectively. ing neutralisation of any disinfectant remaining in the Unfortunately, viable count procedures assume that one sample. Neutralisation or inactivation of residual disinfect- colony develops from one viable cell. Such techniques are, ant can be carried out by dilution, or by the addition of therefore, not ideal for disinfectants (e.g., QACs such as specific agents (see Table 19.3). Using viable counts, it is cetrimide) that promote clumping in bacterial suspensions, possible to calculate the concentration of disinfectant although the problem may be partially mitigated by adding required to kill 99.999% (5-­log kill) of the original suspen- non-­ionic surface-­active agents to the diluting fluid. The sion. Thus, 10 survivors from an original population of 106 European Standards EN 13727:2012+A2:2015 and EN cells represents a 99.999% or 5-­log kill. As bacteria may 13697:2015 provide detailed test procedures for establish- ing the cidal activity of chemical disinfectants against Table 19.3 Neutralising agents for some antimicrobial agents. microbial suspensions and on surfaces in simulated envi- ronmental conditions (carrier test), enabling appropriate Antimicrobial agent Neutralising and/or inactivating agent efficacy claims to be made for a particular disinfectant. Alcohols None (dilution) 19.3.2.2 In-­use and Simulated Use Tests Alcohol-­based Tween 80, saponin, histidine and Apart from suspension tests, in-use testing of used medical hand gels lecithin devices and simulated use tests (involving instruments or Amoxicillin β-­Lactamase from Bacillus cereusa surfaces deliberately contaminated with an organic load Antibiotics (most) None (dilution, membrane filtrationb, and the appropriate test microorganism) have been incor- resin adsorptionc) porated into disinfectant testing protocols. An example is Benzoic acid Dilution or Tween 80d the in-­use test first reported by Maurer in 1972. Here, a dis- Benzylpenicillin β-­Lactamase from B. cereus infectant currently in use in which potentially contami- Bronopol Cysteine hydrochloride nated material (e.g., lavatory brushes, mops) has been placed is tested to see if it contains living microorganisms, Chlorhexidine Lubrol We and egg lecithin or Tween 80 and lecithin (Letheen) and in what numbers. A small volume of fluid is with- drawn from the in-­use container, neutralised in a large vol- Formaldehyde Ammonium ions ume of a suitable diluent and viable counts are performed Glutaraldehyde Glycine on the resulting suspension. Two plates are involved in Halogens Sodium thiosulphate viable count investigations, one of which is incubated for Hexachlorophane Tween 80 3 days at 32 °C (rather than 37 °C, as bacteria damaged by Mercurials Thioglycolic acid (—SH compounds) disinfectants recover more rapidly at lowered tempera- Phenolics Dilution or Tween 80 tures), and the other for 7 days at room temperature. QACs Lubrol W and lecithin or Tween 80 Growth of one or two colonies per plate can be ignored (a and lecithin (Letheen) disinfectant is not usually a sterilant), but 10 or more colo- Sulphonamides p-­Aminobenzoic acid nies would suggest poor and unsatisfactory cidal action. a Simulated use tests involve deliberate contamination of Other appropriate enzymes can be considered, e.g., inactivating or modifying enzymes for chloramphenicol and aminoglycosides, instruments, inanimate surfaces or even skin surfaces, respectively. with a microbial suspension. This may either be under b Filter microorganisms on to membrane, wash, transfer membrane clean conditions or may utilise a diluent containing organic to growth medium. c material (e.g., albumin) to simulate dirty conditions. After Resins for the absorption of antibiotics from fluids are available. d Tween 80 (polysorbate 80). being left to dry, the contaminated surface is exposed to the e Polyethylene glycol ether W–1. test disinfectant for an appropriate time interval. The 374 19 Laboratory Evaluation of Antimicrobial Agents microbes are then removed (e.g., by rubbing with a sterile bacteria survive in nature as intracellular parasites of other swab), resuspended in suitable neutralising medium and microbes, for example, Legionella pneumophila within the assessed for viability as for suspension tests. New products protozoan Acanthamoeba polyphaga. Biocide activity is are often compared with a known comparator compound significantly reduced against intracelluar legionellae (see (e.g., 1-­minute application of 60% v/v 2-­propanol for hand Figure 19.3) and other intracellular pathogens. Disinfectant disinfection products – see European Standard EN1500) to tests involving such bacteria should therefore be conducted show increased efficacy of the novel product. both on planktonic bacteria and on suspensions involving amoebae-­containing bacteria. With the latter, the final bac- 19.3.2.3 Problematic Bacteria terial viable counts are performed after suitable lysis of the Mycobacteria are hydrophobic in nature and, as a result, protozoan host. The legionella/protozoa situation may also exhibit an increased tendency to clump or aggregate in be further complicated by the fact that the microbes often aqueous media. It may be difficult, therefore, to prepare occur as intracellular biofilms. homogeneous suspensions devoid of undue cell clumping (which may contribute to their resistance to chemical dis- 19.3.3 Other Microbe Disinfectant Tests infection). As M. tuberculosis is very slow-­growing, more rapidly growing species such as Mycobacterium terrae, Suspension-­type efficacy tests can also be performed on Mycobacterium bovis or Mycobacterium smegmatis can be other microbes (e.g., fungi and viruses) using similar substituted in tests (as representative of M. tuberculosis). techniques to those described above for bacteria, Recent global public health concerns regarding the increas- although ­significant differences obviously arise in parts ing incidences of tuberculosis (including co-­infections of the tests. with HIV) in developing, low and middle-­income coun- tries (LMICs) and industrialised nations bring into sharp 19.3.3.1 Antifungal (Fungicidal) Tests focus the necessity for representative evaluations of agents In order for disinfectants to claim fungicidal activity, or for with potential tuberculocidal activity. This is particularly the discovery of novel fungicidal agents, a range of stand- true given the high proportion of cases classified as ard tests have been devised. Perhaps the main problem multidrug-­resistant tuberculosis (MDR-­TB). As a result, with fungi concerns the question of which morphological specific standard tests have been developed to permit effec- form of fungus to use as the inoculum. While unicellular tive evaluation of the mycobactericidal activity of biocides yeasts can be treated in much the same manner as bacteria, (see Section 19.3.2). Apart from vegetative bacterial cells, whether to use spores (which may be more resistant than bacterial or fungal spores can also be used as the inoculum the vegetative mycelium) or pieces of hyphae with the fila- in tests. In such cases, incubation of culture plates for the mentous moulds has yet to be fully resolved. Spore suspen- final viability determination should be continued for sev- sions (in saline containing the wetting agent Tween 80) eral days to allow for germination and growth. obtained from 7-­day-­old cultures are presently recom- Compared with suspended (planktonic) cells, bacteria mended. The species to be used may be a known environ- growing on surfaces as biofilms are invariably phenotypi- mental strain and likely contaminant, such as Aspergillus cally more tolerant to antimicrobial agents. With biofilms, niger or Aspergillus brasiliensis, or a pathogen, such as suspension tests can be modified to involve biofilms grown Trichophyton mentagrophytes; other strains such as on coupons of an appropriate glass, metal or polymeric Penicillium variabile are also employed. Clearly, the final substrate, or on the bottom of wells of plastic microtitre selection of organism will vary depending on the perceived plates. After being immersed in, or exposed to, the disin- use for the disinfectant under test. In general, spore sus- fectant solution for the appropriate time interval, the cells pensions of at least 106 CFU/ml have been recommended. from the biofilm are removed, for example, by sonication, Viable counts are typically performed on a suitable medium and resuspended in a suitable microbicide-­neutralising (e.g., malt extract agar, Sabouraud dextrose agar) with medium. Viable counts are then performed on the result- incubation at 20 °C for 48 hours or longer. European ing planktonic cells. A reduction in biomass following anti- Standard EN 1275:2005 specifies that for fungicidal activity microbial challenge can also be monitored using a standard a minimum reduction in viability by a factor of 104 within ­crystal-­violet-­staining technique; however, viable counting 60 minutes is required; test fungi are Candida albicans for permits evaluation of the rate of kill. The Calgary Biofilm yeasticidal activity and C. albicans and A. brasiliensis for Device, discussed in Section 19.9.1, permits the high-­ fungicidal activity. Further procedures may be obtained by throughput screening of antimicrobial agents against reference to European Standards EN 1650:1998 (chemical ­biofilms grown on 96 polycarbonate pegs in a 96-­well micr- disinfectants and antiseptics used in food, industrial, otitre plate. Finally, some important environmental domestic and institutional areas) and EN 13624:2013  19.5 ­Evaluation of Air Disinfectant 375 (disinfectants intended for use in the medical area) and conventional chemical and physical decontamination also AOAC guidance on the fungicidal activity of methods, presenting a unique challenge in infection con- ­disinfectants (955.17). trol. Although numerous published studies on prion ­inactivation by disinfectants are available in the literature, 19.3.3.2 Antiviral (Viricidal) Tests inconsistencies in methodology make direct comparison The evaluation of disinfectants for viricidal activity is a difficult. For example, these variations include: strain complicated process requiring specialised training and ­differences of prion (with respect to sensitivity to thermal facilities; viruses are obligate intracellular parasites and are and chemical inactivation), prion concentration in tissue therefore incapable of independent growth and replication homogenate, exposure conditions and determination of in artificial culture media. They require some other system log reductions from incubation period assays instead employing living host cells. Suggested test viruses include of end-­point titrations. Furthermore, since most studies of rotavirus, adenovirus, poliovirus, herpes simplex viruses, prion inactivation have been conducted with tissue HIV, vaccinia virus, influenza A virus, pox viruses and homogenates, the protective effect of the tissue compo- papovavirus, although extension of this list to include addi- nents may sometimes be significant and thereby contribute tional blood-­borne viruses, such as hepatitis B and hepatitis to resistance to disinfection. Despite this, a reasonable C, significant animal pathogens (e.g., foot and mouth dis- characterisation of effective and ineffective agents has ease virus) and SARS-CoV-2 could be argued, given their emerged and is summarised in Table 19.4. Although most potential impact on public health or the economy of a nation. disinfectants are inadequate for the elimination of prion Briefly, the virus is grown in an appropriate cell line that infectivity, agents such as sodium hydroxide, a phenolic is then mixed with water containing an organic load and formulation, guanidine thiocyanate and chlorine have all the disinfectant under test. After the appropriate time, been shown to have efficacy; further consideration can be residual viral infectivity is determined using a tissue cul- found in Chapters 18 and 20. ture/plaque assay or other system (e.g., animal host or molecular assay for some specific viral component). Such procedures are costly and time-­consuming, and must be 19.4 ­Evaluation of Solid Disinfectants appropriately controlled to exclude factors such as disin- fectant killing of the cell system or test animal. A reduc- Solid disinfectants usually consist of a disinfectant sub- tion of infectivity by a factor of 104 has been regarded as stance diluted by an inert powder. Phenolic substances evidence of acceptable viricidal activity. The European adsorbed onto kieselguhr (diatomite) form the basis of Standard 14476:2013+A2:2019 specifies the minimum many disinfectant powders; another widely used solid dis- standards for viricidal activity of a chemical disinfectant, infectant is sodium dichloroisocyanurate. Other disinfect- and applies to products used in the medical environment, ant or antiseptic powders used in medicine include including surface disinfection by spraying, wiping, acriflavine and compounds with antifungal activity such as ­flooding or other means, textile disinfection, hand sani- zinc undecenoate or salicylic acid mixed with talc. These tiser, hygienic handwashes and instrument disinfection by disinfectants may be evaluated by applying them to suita- immersion. For viruses that cannot be grown in the labo- ble test organisms growing on a solid agar medium. Discs ratory (e.g., hepatitis B), naturally infected cells/tissues may be cut from the agar and subcultured for enumeration must be used. Further test procedures are detailed in of survivors. Inhibitory activity is evaluated by dusting the British Standard BS EN 13610:2002 where the viricidal powders onto the surface of seeded agar plates, using the activity of chemical disinfectants used in food and indus- inert diluents as a control. The extent of growth is then trial areas is evaluated against bacteriophages. The use of observed following incubation. bacteriophage as a model virus in this procedure most likely reflects their ease of growth and survivor enumera- tion via standard plaque assay on host bacterial lawns 19.5 ­Evaluation of Air Disinfectants grown on solid media. The decontamination and disinfection of air are important 19.3.3.3 Prion Disinfection Tests considerations for both infection and contamination con- Prions are a unique class of acellular, proteinaceous infec- trol. A large number of important infectious diseases are tious agent (see Chapter 5), devoid of an agent-­specific spread via microbial contamination of the air. This cross-­ nucleic acid (DNA or RNA). Infection is associated with infection can occur in a variety of situations (hospitals and the abnormal isoform of a host cellular protein called prion care facilities, airplanes, public and institutional build- protein (PrPc). Prions exhibit unusually high resistance to ings), while stringent control of air quality with respect to 376 19 Laboratory Evaluation of Antimicrobial Agents Table 19.4 Efficacy of chemical agents in prion inactivation. be contained in aerosols, including respiratory aerosols, or may occur as airborne particles liberated from some envi- Effective (>3 log10 reduction ronmental source, for example, agitation of spore-­laden Ineffective (≤3 log10 within 1 hour at temperature reduction within 1 hour) 20–55 °C) bed linen or decaying organic matter. Disinfection of air can be carried out by filtration through high-­efficiency Acetone Alkaline detergent (specific formulations) particulate air (HEPA) filters, chemical aerosol/vapour/ fumigation or by ultraviolet germicidal irradiation (UVGI). Alcohol, 50–100% Enzymatic detergent (specific formulation) Increased airflow can aid the distribution of aerosolised Ammonia, 1.0 M Chlorine >1000 ppm disinfectant, and result in dilution of airborne microor- ganisms, thus acting as an adjunct to disinfection. Alkaline detergent (specific Copper, 0.5 mmol/l and H2O2, formulations) 100 mmol/l Although UVGI disinfectant approaches have demon- Beta-­propiolactone Guanidine thiocyanate, >3 M strated efficacy against a range of airborne pathogens and contaminating organisms, it is often more practical to use Chlorine dioxide, 50 ppm Peracetic acid, 0.2% some form of chemical vapour or aerosol to kill them. Ethylene oxide Phenolic disinfectant (specific Formaldehyde vapour is a commonly employed agent for formulation), >0.9% fumigation procedures (not strictly air disinfection), Formaldehyde, 3.7% QAC (specific formulation) although vapourised hydrogen peroxide is increasingly Glutaraldehyde, 5% Hydrogen peroxide, 59% used as an alternative agent. Due to the potential for for- Hydrochloric acid, 1.0 N SDS, 2% and acetic acid, 1% mation of carcinogenic bis(chloromethyl) ether when Hydrogen peroxide, 0.2–60% Sodium hydroxide, ≥1 N used with hydrochloric acid and chlorine-­containing dis- Iodine, 2% Sodium hypochlorite, ≥2% infectants, formaldehyde should not be used in the pres- (20,000 ppm) chlorine ence of hypochlorites. Iodophors Sodium metaperiodate, 0.01 M The work of Robert Koch in the late 1880s demonstrated Ortho-­phthalaldehyde, 0.55% that the numbers of viable bacteria present in air can be Peracetic acid, 0.2–19% assessed by simply exposing plates of solid nutrient media Phenol/phenolics to that air. Indeed, this same process is still exploited in (concentration variable) environmental monitoring in the form of settle plates. Any Potassium permanganate, bacteria that fall on to the plates after a suitable exposure 0.1–0.8% time can then be detected following an appropriate period QAC (specific formulation) of incubation. These gravitational methods are obviously Sodium dodecyl sulphate applicable to many microorganisms, but are unsuitable for (SDS), 1–5% viruses. However, more meaningful data can be obtained if Sodium deoxycholate, 5% force rather than gravity is used to collect airborne parti- Sodium dichloroisocyanurate cles. A stream of air can be directed onto the surface of a nutrient agar plate (surface impaction; slit sampler) or Enzymatic detergent (specific formulations) bubbled through an appropriate buffer or culture medium Triton X-­100, 1% (liquid impingement). Various commercial impactor sam- plers are available. Filtration sampling, where the air is Urea, 4–8 M passed through a porous membrane, which is then cul- Processes may be listed in both columns (ineffective/effective), due to tured, can also be used. For experimental evaluation of different testing parameters or testing methods. All experiments potential air disinfectants, bacterial or fungal airborne ‘sus- conducted without prior cleaning. Source: Adapted from Rutala and Weber (2010). pensions’ can be created in a closed chamber, and then exposed to the disinfectant, which may be in the form of radiation, chemical vapour or aerosol. The airborne micro- airborne contaminants and particulates is critical for con- bial population is then sampled at regular intervals using tamination control in many aseptic procedures. With the an appropriate forced-­air apparatus such as the slit sam- increasing public concern regarding the perceived height- pler. With viruses, the air can be bubbled through a suita- ened threat of bioterrorism, and transmission of pandemic ble liquid medium, which is then subjected to some viral agents including SARS-­CoV-­2, effective air disinfec- appropriate virological assay system. In all cases, problems tion procedures have gained heightened prominence as arise in producing a suitable airborne microbial ‘suspen- potential countermeasures to these significant threats to sion’ and in neutralising residual disinfectant, which may global public health. The microorganisms themselves may remain in the air.  19.7 ­Rapid Evaluation Procedure 377 19.6 ­Evaluation of Preservatives down for injectable and ophthalmic preparations, topical preparations and oral liquid preparations in the British Preservatives are widely employed in the cosmetic and Pharmacopoeia (Appendix XVI C) and the European pharmaceutical industries as well as in a variety of other Pharmacopoeia, which should be consulted for full details of manufacturing industries. The addition of preservatives to the experimental procedures to be used. The United States pharmaceutical formulations to prevent microbial growth Pharmacopeia (USP) specifies an additional category, antac- and subsequent spoilage, to retard product deterioration ids made with aqueous bases, with less-­stringent preservative and to restrain growth of contaminating microorganisms efficacy acceptance criteria, reflecting the unique challenges is commonplace for non-­sterile pharmaceutical formula- of adequately preserving aqueous formulations containing tions as well as low-­volume aseptically prepared formula- significant proportions (in % w/v) of suspended solids and tions intended for multiple use from one container (see high concentrations of divalent cations, Mg2+, Ca2+ and Al3+. Chapter 17). Indeed, adequate preservation (and validation In some instances, the range and/or spectrum of preserva- of effectiveness) is a legal requirement for certain formula- tion can be extended by using more than one preservative at tions. Effective preservation prevents microbial and, as a a time, and a number of synergistic preservative combina- consequence, related chemical, physical and aesthetic tions have been described (see also Chapter 20). Thus, a com- spoilage that could otherwise render the formulation unac- bination of parabens (p-­hydroxybenzoic acid) with varying ceptable for patient use, therapeutically ineffective or water solubilities may protect both the aqueous and oil harmful to the patient (due to the presence of toxic metab- phases of an emulsion, while a combination of Germall 115 olites or microbial toxins). The factors which influence the (an imidazolidinyl urea derivative) and parabens results in a activity of the antimicrobial agent when employed as a pre- preservative system with both antibacterial (Germall 115) servative are largely those which affect disinfectant activity and antifungal (parabens) activities. (described in Section 19.2.4); however, when considering the activity of the agent, the interactions with formulation components (adsorption to suspended particles and oil– 19.7 ­Rapid Evaluation Procedures water partitioning, for instance; see also Chapter 17) should be considered as additional factors which can In most of the tests mentioned above, results are not avail- potentially attenuate the preservative activity. able until visible microbial growth occurs, at least in the While the inhibitory or cidal activity of the preservative controls. This usually takes 24 hours or more. The potential chemical can be evaluated using an appropriate in vitro test benefits of rapid antimicrobial susceptibility screening pro- system (see Sections 19.3.2.1 and 19.8.1.2), its continued cedures are obvious, particularly in aggressive infections or activity when combined with the other ingredients in the rapidly progressing nosocomial infections of immunocom- final manufactured product must be established. Problems promised patients where appropriate antimicrobial selec- clearly exist with some products, where partitioning into tion is critical. To date, only a few rapid methods for various phases may result in the absence of preservative in detecting microbial viability or growth are presently one of the phases, for example, oil-­in-­water emulsions where employed in assessing the efficacy of antimicrobials. For the preservative may partition only into the oily phase, allow- the main, these rapid antimicrobial susceptibility tests ing any contaminant microorganisms to flourish in the aque- (ASTs) have focused on rapid determination of the resist- ous phase. In addition, one or more of the components may ance profile of clinical pathogens, either through genotypic inactivate the preservative. Consequently, suitably designed means (looking for the presence of specific resistance simulated use challenge tests involving the final product are, genes) or by phenotypic methods (including microscopy-­ therefore, required in addition to direct potency testing of the based direct observation of response to antibiotics, or pure preservative. In the challenge test, the final preserved microscopy paired with microfluidics or other optical tech- product is deliberately inoculated with a suitable environ- niques). These latter approaches include epifluorescent mental microorganism which may be fungal (e.g., C. albicans and bioluminescence techniques. The former relies on the or A. brasiliensis) or bacterial (e.g., S. aureus, E. coli, P. aerugi- fact that when exposed to the vital stain acridine orange nosa). For oral preparations with a high sucrose content, the and viewed under UV light, viable cells fluoresce green or British and European Pharmacopoeias include the osmo- greenish yellow, while dead cells appear orange. Live/dead philic yeast Zygosaccharomyces rouxii as a recommended staining of sessile bacterial populations has the potential to challenge organism. The subsequent survival (inhibition), yield important data with respect to antimicrobial suscepti- death or growth of the inoculum is then assessed using viable bility, but requires skilled personnel and specialised count techniques. Different performance criteria are laid microscopy equipment. 378 19 Laboratory Evaluation of Antimicrobial Agents With tests involving liquid systems, the early growth of (antibiotics) invariably have determination of MIC as their viable cells can be assessed by some light-­scattering pro- main focus. Tests for the bacteriostatic activity of antimi- cesses; blood culture techniques have classically used the crobial agents are valuable tools in predicting antimicro- production of CO2 as an indicator of bacterial metabolism bial sensitivity/tolerance in individual patient samples and and growth. In addition, the availability of molecular tech- for detection and monitoring of resistant bacteria. However, niques, such as quantitative polymerase chain reaction correlation between MIC and therapeutic outcome is (PCR), may be useful in demonstrating the presence or ­frequently difficult to predict, especially in chronic biofilm-­ growth of microorganisms that are slow or difficult to cul- mediated infections. The determination of MIC values ture under usual laboratory conditions, for example, must be conducted under standardised conditions, since viruses. This may obviate the need to neutralise residual deviation from standard test conditions can result in con- disinfectant with some assays. siderable variation in data. Rapid colourimetric assays for antimicrobial susceptibility have been developed including the commercially available 19.8.1 Tests for Bacteriostatic Activity VITEK2 system (BioMérieux) and colourimetric tests based on the extracellular reduction of tetrazolium salts 2-­(2-­meth The historical gradient-­plate, ditch-­plate and cup-­plate o x y - 4­ - ­n i t r o p h e n y l ) - ­3 - ­( 4 - ­n i t r o p h e n y l ) - ­5 - ­( 2 , 4 -­ techniques (see earlier editions of this book) have been disulphophenyl)-­2H-­tetrazolium monosodium (WST-­8) and replaced by more quantitative techniques such as disc dif- 2,3-­bis[2-­methoxy-­4-­nitro-­5-­sulphophenyl]-­2H-­tetrazolium-­ fusion (Figure 19.4), broth and agar dilution and E-­tests 5-­carboxanilide (XTT). These latter studies have demon- (Figure 19.5). All employ chemically defined media (e.g., strated the potential for the tetrazolium salts WST-­8 and Mueller–Hinton or Iso-­Sensitest) at a pH of 7.2–7.4, and, in XTT to be used in the rapid, accurate and facile screening of the case of solid media, agar plates of defined thickness. antimicrobial susceptibility and MIC determination in a Regularly updated guidelines are provided by the Clinical range of bacteria, including staphylococci, extended β-­ & Laboratory Standards Institute (CLSI) (formerly the lactamase-­producing clinical isolates (E. coli, Enterococcus National Committee for Clinical Laboratory Standards faecalis) and P. aeruginosa. Using this method, MIC values [NCCLS]) and are widely used in many countries; histori- in agreement with those obtained using standard methods cally, the British Society for Antimicrobial Chemotherapy can be obtained after just 5 hours. The application of matrix-­ assisted laser desorption ionisation-­time-­of-­flight mass spec- trometry (MALDI-­TOF MS) has also become commonplace in clinical microbiology, and progress has been made in the use of this powerful technique not only in the rapid identifi- cation of clinically significant microorganisms, but in deter- mination of antimicrobial susceptibility/resistance profiles. In addition, signal amplification techniques employing time-­resolved fluorescence or nucleic acid amplification technology (NAAT) have been described; however, the requirement for specific equipment and training, alongside the necessity to compare with real-­time phenotypic charac- terisation, may limit the utility of such techniques in rou- tine clinical microbiology laboratories. In general, however, approaches described in this section for rapid AST are of potential value in the rapid validation of microbicidal/dis- infectant activity, but are not currently regarded as, or incorporated into, standard disinfectant methods. Figure 19.4 Disc test with inhibition zones around two (1, 2) of 19.8 ­Evaluation of Potential five discs. The zone around disc 1 is clear and easy to measure, Chemotherapeutic Antimicrobials whereas that around disc 2 is indistinct. Although none of the antimicrobials in discs 3, 4 or 5 appear to inhibit the bacterium, synergy (as evidenced by inhibition of growth between the discs) Unlike tests for the evaluation of disinfectants, where is evident with the antimicrobials in discs 3 and 5. Slight determination of cidal activity is of paramount impor- antagonism of the drug in disc 1 by that in disc 3 is evident by tance, tests involving potential chemotherapeutic agents the slightly reduced zone of inhibition in proximity to disc 3.  19.8 ­Evaluation of Potential Chemotherapeutic Antimicrobial 379 qualitative; however, the diameter of the zone of inhibi- tion may be correlated to MIC determination through a linear regression analysis (Figure 19.6). Although subtle variations of the disc test are used in some countries, the basic principles behind the tests remain similar, and are based on the original work of Bauer and colleagues (Kirby–Bauer method). Some tech- niques employ a control bacterial isolate on each plate so that comparisons between zone sizes around the test and control bacterium can be ascertained (i.e., a disc potency control). Provided that discs are maintained and handled as recommended by the manufacturer, the value of such controls becomes debatable and probably unnecessary. Control strains of bacteria are available which should have inhibition zones of a given diameter with stipulated anti- microbial discs. Use of such controls endorses the suitabil- ity of the methods (e.g., medium, inoculum density, incubation conditions) employed. For slow-­growing Figure 19.5 E-­test on an isolate of Candida albicans. Inhibition zone edges are distinct and the MICs for itraconazole (IT) and microorganisms, the incubation period can be extended. fluconazole (FL) (0.064 and 1.5 mg l–1, respectively) are easily Problems arise with disc tests where the inoculum density decipherable. is inappropriate (e.g., too low, resulting in an indistinct edge to the inhibition zone following incubation), or has produced its own guidelines and testing procedures where the edge is obscured by the sporadic growth of cells (e.g., see Howe and Andrews 2012), but has in recent years within the inhibition zone, that is, the initial inoculum played an essential role in the European Committee on although pure contains cells expressing varying levels of Antimicrobial Susceptibility Testing (EUCAST) to harmo- susceptibility – so-­called heterogeneity. As the distance nise break-­point data, develop methodology and standard- from the disc increases, there is a logarithmic reduction in ise susceptibility testing throughout Europe. the antimicrobial concentration; the result is that small differences in zone diameter with antimicrobials (e.g., 19.8.1.1 Disc Tests vancomycin) which diffuse poorly through solid media These are really modifications of the earlier cup-­or ditch-­ may represent significantly different MICs. Possible syner- plate procedures where filter-­paper discs impregnated gistic or antagonistic combinations of antimicrobials can with the antimicrobial replace the antimicrobial-­filled often be detected using disc tests (Figure 19.4). cups or wells. For disc tests, standard suspensions (e.g., 0.5 McFarland standard – this is a measure of the concen- 19.8.1.2 Dilution Tests tration of a microbial population based on optical density) These usually employ liquid media but can be modified to of cells harvested in the log-­phase of growth are prepared use solid media. Doubling dilutions, usually in the range and inoculated on to the surface of appropriate agar plates 0.008–256 mg l–1 of the antimicrobial under test, are pre- to form a lawn. Commercially available filter-­paper discs pared in a suitable broth medium and an aliquot of log-­ containing known concentrations of antimicrobial agent phase cells is added to each dilution to result in a final cell (it is possible to prepare your own discs for use with novel density of around 5 × 105 CFU ml–1. After incubation at drugs) are then placed on the dried lawn and the plates are 35 °C for 18 hours, the concentration of antimicrobial con- incubated aerobically at 35 °C for 18 hours. The density of tained in the first clear tube is read as the MIC. Needless to bacteria inoculated on to the plate should produce just say, dilution tests require a number of controls, for exam- confluent growth after incubation for that period. Any ple, sterility control, growth control and the simultaneous zone of inhibition occurring around the disc is then meas- testing of a bacterial strain with known MIC to show that ured and, after comparison with known standards, the the dilution series is correct. End points with dilution tests bacterium under test is identified as susceptible or resist- are usually sharp and easily defined, although ‘skipped’ ant to that particular antibiotic. For novel agents, these wells (inhibition in a well with growth either side) and sensitivity parameters are only available after extensive ‘trailing’ (a gradual reduction in growth over a series of clinical investigations have been correlated with wells) may be encountered. The latter is especially evident laboratory-­generated data. Disc tests are basically with antifungal tests (see below). Nowadays, the dilution 380 19 Laboratory Evaluation of Antimicrobial Agents 2 Figure 19.6 A scattergram and regression line ≥264.0 analysis correlating zone diameters and MICs. The break points of susceptible (MIC ≤ 2.0 mg l–1, zone diameter ≥ 21 mm) and resistant (MIC ≥ 8.0 mg l–1, 32.0 C zone ≤ 15 mm) bacteria are shown by the dotted lines. For a complete correlation between 16.0 MICs and zone diameter, all susceptible, intermediate and resistant isolates should fall in boxes A, B and C, respectively. Errors (correlations 3 2 3 3 3 8.0 outside these boxes) occur. Source: Courtesy of B Dr. Z. Hashmi. 3 7 11 12 2 3 2 2 4.0 MIC (mg l–1) 3 2 3 3 2 2 6 3 2.0 A 2 7 10 15 11 5 3 2 2 1.0 2 3 8 16 17 21 13 11 10 2 2 2 0.5