Sensitivity of Microorganisms

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Chapter: Pharmaceutical Microbiology : Sterilization Procedures And Sterility Assurance

The general pattern of resistance of microorganisms to biocidal sterilization processes is independent of the type of agent employed (heat, radiation or gas), with vegetative forms of bacteria and fungi, along with the larger viruses, showing a greater sensitivity to sterilization processes than small viruses and bacterial or fungal spores.



The general pattern of resistance of microorganisms to biocidal sterilization processes is independent of the type of agent employed (heat, radiation or gas), with vegetative forms of bacteria and fungi, along with the larger viruses, showing a greater sensitivity to sterilization processes than small viruses and bacterial or fungal spores. The choice of suitable reference organisms for testing the efficiency of sterilization processes is therefore made from the most durable bacterial spores; these are usually represented by Bacillus stearothermophilus for moist heat, certain strains of B.subtilis for dry heat and gaseous sterilization, and B.pumilus for ionizing radiation.


Ideally, when considering the level of treatment necessary to achieve sterility a knowledge of the type and total number of microorganisms present in a product, together with their likely response to the proposed treatment, is necessary. Without this information, however, it is usually assumed that organisms within the load are no more resistant than the reference spores or than specific resistant product isolates. In the latter case, it must be remembered that resistance may be altered or lost entirely by repeated laboratory subculture and the resistance characteristics of the maintained strain must be regularly checked.


A sterilization process may thus be developed without a full microbiological background to the product, instead being based on the ability to deal with a ‘worst case’ condition. This is indeed the situation for official sterilization methods, which must be capable of general application, and modern pharmacopoeial recommendations are derived from a careful analysis of experimental data on bacterial spore survival following treatments with heat, ionizing radiation or gas.


However, the infectious agents responsible for spongiform encephalopathies (prions) such as bovine spongiform encephalopathy (BSE) and Creutzfeldt –Jakob disease (CJD) exhibit exceptional degrees of resistance to many lethal agents. Recent work has even cast doubt on the adequacy of the process of 18 minute exposure to steam at 134–138°C which has been recommended for the destruction of prions (and which far exceeds the lethal treatment required to achieve adequate destruction of bacterial spores).


a)   Survivor Curves


When exposed to a killing process, populations of micro-organisms generally lose their viability in an exponential fashion, independent of the initial number of organisms. This can be represented graphically with a ‘survivor curve’ drawn from a plot of the logarithm of the fraction of survivors against the exposure time or dose (Figure 21.1). Of the typical curves obtained, all have a linear portion which may be continuous (plot A), or may be modified by an initial shoulder (B) or by a reduced rate of kill at low survivor levels (C). Furthermore, a short activation phase, representing an initial increase in viable count, may be seen during the heat treatment of certain bacterial spores. Survivor curves have been employed principally in the examination of heat sterilization methods, but can equally well be applied to any biocidal process.


b)   Expressions Of Resistance


i)  D-value


The resistance of an organism to a sterilizing agent can be described by means of the D-value. For heat and radiation treatments, respectively, this is defined as the time taken at a fixed temperature or the radiation dose required to achieve a 90% reduction in viable cells (i.e. a 1 log cycle reduction in survivors; Figure 21.2A). The calculation of the D-value assumes a linear type A survivor curve (Figure 21.1), and must be corrected to allow for any deviation from linearity with type B or C curves. Some typical D-values for resistant bacterial spores are given in Table 21.1.


ii)  Z-value


For heat treatment, a D-value only refers to the resistance of a microorganism at a particular temperature. In order to assess the influence of temperature changes on thermal resistance, a relationship between temperature and log D-value can be developed, leading to the expression of a Z-value, which represents the increase in temperature needed to reduce the D-value of an organism by 90% (i.e. 1 log cycle reduction; Figure 21.2 B). For bacterial spores used as biological indicators for moist heat (B. stearothermophilus) and dry heat (B. subtilis) sterilization processes, mean Z-values are given as 10 °C and 22 °C, respectively. The Z-value is not truly independent of temperature but may be considered essentially constant over the temperature ranges used in heat sterilization processes.


c)    Sterility Assurance


The term ‘sterile’, in a microbiological context, means no surviving organisms whatsoever. Thus, there are no degrees of sterility; an item is either sterile or it is not, and so there are no levels of contamination which may be considered negligible or insignificant and therefore acceptable. From the survivor curves presented, it can be seen that the elimination of viable microorganisms from a product is a time-dependent process, and will be influenced by the rate and duration of biocidal action and the initial microbial contamination level. It is also evident from Figure 21.2 A that true sterility, represented by zero survivors, can only be achieved after an infinite exposure period or radiation dose. Clearly, then, it is illogical to claim, or expect, that a sterilization procedure will guarantee sterility. Thus, the likelihood of a product being produced free of microorganisms is best expressed in terms of the probability of an organism surviving the treatment process, a possibility not entertained in the absolute term ‘sterile’. From this approach has arisen the concept of sterility assurance or a microbial safety index which gives a numerical value to the probability of a single surviving organism remaining to contaminate a processed product. For pharmaceutical products, the most frequently applied standard is that the probability, post-sterilization, of a non-sterile unit is no more than 1 in 1 million units processed (i.e. ≤ 10− 6).The sterilization protocol necessary to achieve this with any given organism of known D-value can be established from the inactivation factor (IF) which may be defined as: 

IF =10t̷D

where t is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing radiation) and D is the D-value appropriate to the process employed.


Thus, for an initial burden of 102 spores an inactivation factor of 108 will be needed to give the required sterility assurance of 10−6 (Figure 21.3). The sterilization process will therefore need to produce sufficient lethality to achieve an 8 log cycle reduction in viable organisms; this will require exposure of the product to eight times the D-value of the reference organism (8 D). In practice, it is generally assumed that the contaminant will have the same resistance as the relevant biological indicator spores unless full microbiological data are available to indicate otherwise. The inactivation factors associated with certain sterilization protocols and their biological indicator organisms are given in Table 21.1.


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