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Chapter: Pharmaceutical Microbiology : Laboratory Evaluation Of Antimicrobial Agents

A biocide may be defined as a chemical or physical agent which kills viable organisms, both pathogenic and non-pathogenic. This broad definition clearly includes microoganisms, but is not restricted to them.




Key terms such as disinfection, preservation, antisepsis and sterilization can be seen in upcoming articles. A number of other important terms used to describe the antimicrobial activity of agents are also commonly used. A biocide may be defined as a chemical or physical agent which kills viable organisms, both pathogenic and non-pathogenic. This broad definition clearly includes microoganisms, but is not restricted to them. The term microbicide is therefore also used to refer specifically to an agent which kills microorganisms (germicide may also be used in this context, but generally refers to pathogenic microorganisms). The terms biocidal, bactericidal, fungicidal and viricidal therefore describe an agent with killing activity against a specific class or classes of organism indicated by the prefix, whereas the terms bacteriostatic and fungistatic refer to agents which inhibit the growth of bacteria or fungi (Figure 18.1), but do not necessarily kill them. It should be noted, however, that some microorganisms that appear non-viable and non-cultivable fol-lowing antimicrobial challenge may be revived by appropriate methods, and that organisms incapable of multiplication may retain some enzymatic activity.


In the laboratory evaluation of antibacterial agents, the terms minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are most commonly used. Recently published British Society for Chemotherapy (BSAC) guidelines for the determination of minimum inhibitory concentrations (see Further Reading) define the MIC as the lowest concentration of antimicrobial which will inhibit the visible growth of a microorganism after overnight cultivation and the MBC as the lowest concentration of antimicrobial that will prevent the growth of a microorganism after subculture onto antibiotic-free media. Generally, MIC and MBC values are recorded in milligrams per litre or per millilitre (mg/L or mg/ml). With most cidal antimicrobials, the MIC and MBC are frequently near or equal in value, although with essentially static agents (e.g. tetracycline), the lowest concentration required to kill the microorganism (i.e. the MBC) is invariably many times the MIC and often clinically unachievable without damage to the human host. As with microbicides, cidal terms can be applied to studies involving not just bacteria but other microbes, e.g. when referring to cidal antifungal agents the term minimum fungicidal concentration (MFC) is used. Recently, thanks to developments in the design of high-throughput laboratory screens for biofilm susceptibility, the minimum biofilm eradication concentration (MBEC) can be accurately determined for organisms grown as single or mixed species biofilms. The MBEC is the minimum concentration of an antimicrobial agent required to kill a microbial biofilm. For conventional antibiotics and biocides the MBEC value may be 1000-fold higher than the MBC value for the same planktonic microorganisms. Further studies have shown that often no correlation exists between the MIC and the MBEC, indicating the potential limitations of therapeutic antibiotic selection based on determined MIC values.


The term tolerance implies the ability of some bacterial strains to survive (without using or expressing resistance mechanisms), but not grow, at levels of antimicrobial agent that should normally be cidal. This applies particularly to systems employing the cell-wall-active β-lactams and glycopeptides, and to Gram-positive bacteria such as streptococci. Normally, MIC and MBC levels in such tests should be similar (i.e. within one or two doubling dilutions); if the MIC/MBC ratio is 32 or greater, the term tolerance is used. Tolerance may in some way be related to the Eagle phenomenon (paradoxical effect), where increasing concentrations of antimicrobial result in reduced killing rather than the increase in cidal activity expected (see Figure 18.2). Tolerance to elevated antimicrobial challenge concentrations is also a characteristic of microbial biofilm populations. Finally, the term resistance has several definitions within the literature, however, it generally refers to the ability of a microorganism to withstand the effects of a harmful chemical agent, with the organism neither killed nor inhibited at concentrations to which the majority of strains of that organism are susceptible. Resistance mechanisms generally involve modification of the normal target of the antimicrobial agent either by mutation, enzymatic changes, target substitution, antibiotic destruction or alteration, antibiotic efflux mechanisms and restricted permeability to antibiotics.


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