Rotation of Disinfectants to Combat Microbial Resistance
Regulatory and advisory groups have recommended disinfectant rotation be practiced as part of a comprehensive sanitation program. Here`s why.
By Elaine M. Kopis
As early as 1911, scientists were speculating that the onset of microbial resistance to both drugs and disinfectants would one day be a serious concern to the infection control community. In recent years, this speculation has become fact
due to observations of a variety of gram negative and gram positive bacteria with demonstrated resistance to traditional chemotherapeutics. The pharmaceutical contamination control industry has also speculated about the existence of microorganisms which appear to exhibit resistance to commonly and frequently used chemical disinfectants. A great deal has been written regarding the resistance of certain microorganisms to a variety of chemical disinfectants, including quaternary ammonium chloride and phenolic compounds. Obviously, the phenomenon of microbial resistance is of considerable concern to pharmaceutical manufacturers, who must rely upon frequent cleaning and disinfecting to maintain the integrity of aseptic filling areas. This phenomenon has lead to the recommendation by both regulatory and advisory groups [1, 2] that disinfectant rotation be practiced as part of a comprehensive sanitation program for cleanrooms.
The phenomenon of resistance may be defined as: the inability of a microorganism to be killed by an established disinfectant with a demonstrated efficacy against a similar strain at a specified concentration. Resistance may be naturally occurring within a particular species or strain, or acquired. Acquired resistance may occur through mutations in sensitive cell populations or through a process known as “transference,” that results in transferring of genetic material from one organism to another. This transference of DNA may lead to the transfer of resistance genes within or even between species. This genetically based resistance has been demonstrated with Enterobacteriaceae against phenolic compounds . Examples of acquired resistance are also available. One such study observed resistance in Serratia marcescens  against quaternary ammonium chloride compounds.
Although microbial resistance to various disinfectants has been observed, methods by which this resistance may be countered are few and are not well established empirically. However, two recent studies involving the use of phenolic disinfectants against Pseudomonas aeruginosa demonstrate that a sanitizing plan involving the use of an alkaline and an acidic disinfectant in rotation enhanced the overall effectiveness of the disinfectants.
The first study  compares the efficacies of two phenolic disinfectants, one acidic and the other alkaline, against P. aeruginosa when applied on a rotational versus a continuous basis under defined laboratory conditions. P. aeruginosa (ATCC 15442) was chosen for this study because of its intrinsic resistance to phenolic compounds. The two phenolic products were chosen based on their chemical compatibility and efficacy profiles. During this study, inocula of this organism were evenly dispersed on agar surfaces. Following a suitable drying period, a paper sensi-disc was placed on the center of each agar surface and treated with a use-dilution of the disinfectant. The plate was then incubated at 30°C for 48 hours in an inverted position. (A water control was included in this study.) Surviving cells which were identified to be within the periphery of the zone of inhibition were subcultured and then the process repeated. The protocol compared the efficacies of the use-dilutions of the alkaline and acidic phenolic disinfectants when applied continuously and when applied in rotation with each other. The water control included in this study predictably yielded no zone of inhibition. In all cases, the efficacy of the disinfectant was determined by the size of the zone of inhibition. A total of 40 transfers per treatment for each of the two trials conducted were performed.
The data from both trials show that after several transfers, only the zone of inhibition produced by treatment with the alkaline disinfectant alone decreased in size. The data obtained from these observations indicate that the P. aeruginosa culture displayed signs of resistance to the alkaline phenolic disinfectant. Data from the trials involving the use of the acidic phenolic disinfectant were not conclusive. Trial One showed some adaptation of the P. aeruginosa; however, trial two showed small numbers of suspect resistant colonies which upon transfer did not result in a reduced zone.
The trials where the alkaline phenolic application was rotated with the acidic phenolic application showed no signs of adaptation or developed resistance, because the size of the zones of inhibition did not decrease from subsequent transfers and treatments.
The second study  compared the efficacies of the same two phenolic disinfectants against a well-est a blished population of adherent Pseudomonas aeruginosa on a stainless steel surface. This study is probably of more significance to pharmaceutical manufacturers because bacterial biofilms are more difficult to remove than common bioburden.
In this study, inocula of P. aeruginosa ATCC 15442 were utilized to coat sterile stainless steel coupons via a sterile cotton swab. Based on results of RODAC testing, the coupons were inoculated 13 consecutive times in order to create a well-established adherent population of greater than 350 colonies/10 cm2. The coupons were then treated with water as a control, the continuous application of a use-dilution of an alkaline phenolic disinfectant, the continuous application of a use-dilution of an acidic phenolic disinfectant, and the rotational application of the alkaline and the acidic phenolic disinfectants.
The treatment involved dipping the coupons into the disinfectant solutions, followed by a sterile water dip. The coupons were allowed to drain and then returned to sterile glass petri dishes, where they were held at room temperature. Twenty-four hours after treatment, the coupons were sampled utilizing RODAC plates to enumerate surviving organisms, and immediately reinoculated to maintain the well-established colonies, as stated above. Following another 24 hours of incubation, the disinfectant treatment, followed by sampling and reinoculationwas repeated for a total of 24 consecutive treatments.
The results of the study show that throughout 24 consecutive treatments, the alkaline phenolic disinfectant failed to inactivate the P. aeruginosa to a significant degree. The acidic phenolic disinfectant showed a reduction in recovery. However, when each disinfectant was used in a continuous application, as described above, the P. aeruginosa persisted. However, the application of the two disinfectants in rotation showed inactivation of the organism. The improvement in activity against P. aeruginosa was observed after individual treatments utilizing both the alkaline and the acidic disinfectant when the rotational approach was taken.
The mode of action which results in the inactivation of the adherent organisms through a rotational application has not yet been well-defined. However, speculation suggests extremes in pH, contributed by the use of compatible alkaline and acidic phenolic disinfectants, augmented the ability of these antimicrobial agents to penetrate the surface-adherent bacteria. This study represents the strongest evidence thus far supporting a rotational approach to hard surface disinfection. n
1. “Annex on the Manufacture of Sterile Medicinal Products; Revision of the Guide to Good Manufacturing Practice,” Working Party on “Control of Medicines and Inspections,” European Commission, June 1995 (section 37).
2. ISO/CD 13408.3 Aseptic Processing of Health Care Products, section 11, Cleaning and Disinfection of the APA, (18).
3. Bauernfeind, A., Burrows, R.R., and Petermüller, C., “Genetically Determined Changes in Sensitivity to Components of Disinfectants or Enterobacteriaceae,” Hyg. Med. 6, 1981 (310-313).
4. Chaplin, C.E., “Bacterial Resistance to Quaternary Ammonium Disinfectants,” Journal of Bacteriology 63, 1952 (453-458).
5. Conner, D.E., Eckman, M.K., “Rotation of Phenolic Disinfectants,” Pharmaceutical Technology, Sept. 1992 (148-160).
6. Conner, D.E., Eckman, M.K., “Rotation of Phenolic Disinfectants Enhances Efficacy Against Adherent Pseudomonas aeruginosa,” Pharmaceutical Technology,Oct.1993 (94-104).
Elaine Kopis received her BS degree in Chemistry from Southern Illinois University. Her current position is Technical Service Chemist for the ConvaTec Contamination Control Group. In this position, her focus area is microbial control in critical environments. Prior to her current position, Kopis held the title of Associate Manager of Quality Control with ConvaTec/Calgon Vestal for five years. In this position, she was responsible for the daily management of both the Analytical and Microbiological testing laboratories. Prior to moving to Quality Control, Kopis was a Technical Service Chemist in Calgon Vestal`s Infection Control Group for two years. Before joining ConvaTec/Calgon Vestal, she was an Application Engineer with the Nalco Chemical Company. Kopis is a member of the Association of Official Analytical Chemists, the American Chemical Society, the American Society for Quality Control, the Parenteral Drug Association, and the CleanRooms Editorial Advisory Board.