I HAVE MODERATED THE PMF LIST, an email discussion list on the Internet dedicated to topics of interest to microbiologists in the pharmaceutical and personal care industries; a list that has been active since 1996 (http: //www.microbiol.org/pmflist_info.htm). I mention this only because in that time I have had the opportunity to develop a perspective on issues that interest the working microbiologist. Occasionally, the odd topic will really catch fire in the discussion group. This happened recently in response to an interesting article that appeared in the March 2005 issue of A2C2 Magazine [1] dealing with the advisability of disinfectant rotation. In this thoughtful article, the author argued for the need to “rotate” a disinfectant with a sporicide. The interest in this topic initially came as a surprise to me, as I had thought the issue long since resolved in the pharmaceutical and personal care industries.

As the discussion continued on the PMF List, the points of contention became more clear. A great deal of discussion revolved around the concept that a microorganism could become resistant to a disinfectant. Here is where the first bit of clarification is required. A microorganism will not become resistant to much of anything. It either is or it is not affected by the compound. Within a very large population of microorganisms, there is a chance (normally a chance of approximately 10-6) that a cell within the population will have a mutation at a specific gene. This might provide some competitive advantage under some environments, perhaps survival in the presence of an elevated level of a chemical. The mutation that promotes survival under these conditions could, over the course of a few generations, become the dominant genotype in that population. This is, of course, assuming that the challenge is not too great. For example, it does not matter how acidotolerant a particular mutant is, growth in 6N hydrocholoric acid is just plain unlikely—the conditions are too inhospitable to life.

Can a low level of exposure to biocides select variants in a population that are more tolerant than the previous dominant type? Can low level exposure select “resistant” microorganisms? Absolutely The phenomenon of biocide resistance is well known and has been extensively reviewed in the literature [2-5]. Extensive work on the mechanism

of action allowing previously susceptible microorganisms to survive elevated levels of the biocide have highlighted efflux pumps as a major contributor to the biocide resistance [6,7], as well as physiological adaptation [8].

The next question is one of significance. Is this phenomenon of any consequence to the pharmaceutical cleanroom sanitization program? Well, that is what I hope to answer in this short essay.

The Development of Resistance in Bacterial Populations Genetic Resistance

The literature provides many examples of microorganisms able to survive in disinfectants. This can be either in laboratory experiments using an increasing level of biocide to select variants in the population, or by examination of biocidal solutions for the presence of resistant microorganisms. The gram-negative bacilli are the most frequent isolates from this type of evaluation [9-15]. This may be due to a combination of causes including alterations in outer membrane permeability due to changes in porin diameters [16-19]. In addition, it is not clear that the outer-membrane mediated resistance is in fact due to selection of a mutant genotype from the population, or rather phenotypic adaptation, as this trait has been reported to be rapidy lost once the selective pressure is removed.

The other type of experiment performed to demonstrate development of genetic resistance is one in which a laboratory researcher takes a microorganism in culture and exposes it to increasing levels of biocides, selecting resistance variants at each stage. There is one such set of experiments of particular relevance to the pharmaceutical microbiologist that we will examine in some detail as an example.