IMIDAZOLIDINYL UREA RESISTANT PSEUDOMONAS 421 1 , .• 0 4 24 48 72 1 68 556 Hourm Figure 7. Growth of P. cepacia 445 in emulsion preserved with 0.5% imidazolidinyl urea ([•) and 0.5% imidazolidinyl urea plus methyl paraben 0.3% (--), 0.5% (0), or 1.0% (/•). amount of imidazolidinyl urea remaining after growth in preserved broth was deter- mined by a colorimetric assay. The percent decrease in imidazolidinyl urea content of the broth flitrate was calculated (Table III). The presence of the resistant strains P5,445, RC, and 495 resulted in an 80% decrease in the concentration of imidazolidinyl urea. Significantly higher concentrations of the imidazolidinyl urea were recovered from the broth inoculated with the non-resistant strain, ATCC 9027. Those organisms able to grow in the presence of high concentra- tions of imidazolidinyl urea contained low levels of the preservative in the broth flitrate. DISCUSSION This study has demonstrated the resistance of in-house product isolates to imidazoli- dinyl urea. It was hypothesized that perhaps by repeated exposure to sublethal concen- trations of imidazolidinyl urea, these organisms began to selectively develop resistance to that compound. The resistance is specific for imidazolidinyl urea and does not cross react with other classes of preservatives. The results of the spectrophotometric assay for imidazolidinyl urea indicate that these organisms may be able to metabolize or chemi- cally inactivate the imidazolidinyl urea. Future testing will include analysis of the filter disks after filtration to detect imidazolidinyl urea which may have been bound to the bacterial cells. Because the resistance for most of these organisms is not evident in the first 24 hours of testing, using D-values and linear regression analysis to predict preservative efficacy against these organisms after just 24 hours of testing could give the microbiologist a

422 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table III Imidazolidinyl Urea Concentration After Incubation of Inoculated Broth for 21 Days at 25øC IU Conc. Decrease IU Conc. Decrease IU Conc. Decrease Organism (g/100 g) (%) (g/100 g) (%) (g/100 g) (%) P5 0.07 • 85 0.11 b 88 0.27 c 85 445 0.09 81 0.08 91 0.11 94 495 0.07 85 0.12 87 0.29 84 RC 0.05 89 0.08 91 0.14 92 9027 0.42 12 0.74 19 1.6 11 Uninoculated Control 0.48 0 0.91 0 1.8 0 Initial concentration was 0.48 g IU/100 g. Initial concentration was 0.91 g IU/100 g. Initial concentration was 1.8 g IU/100 g. false sense of security. Therefore, because of the lag period required before resistance is demonstrated, a longer challenge test procedure is recommended. An increasing number of these resistant organisms are being recovered in the manufac- turing environment. One source of these adapted organisms can be improperly cleaned equipment. If dilute product remains on mixing vessels, or in hoses or pipes, microor- ganisms which may be present on the equipment can adapt to the diluted preservatives, thereby producing a resistant population of organisms. In conclusion, cosmetic microbiologists must monitor the organisms recovered from the manufacturing environment for preservative resistance. Utilization of multiple preser- vative systems will minimize increased organism resistance to one preservative, but additional precautions in manufacturing for adequate cleaning and sanitizing will help eliminate organisms and prevent their exposure to sublethal levels of preservative. ACKNOWLEDGEMENTS We wish to express our appreciation to James C. Garey of the Analytical Department for the spectrophotometric studies reported here, and to Mary Ann Wichmann for her participation in these research studies. REFERENCES (1) F. W. Adair, S. G. Geftic, and J. Gelzer, Resistance of Pseudomonas to quaternary ammonium com- pounds, Applied Microbiology, 21, 1058-1063 (1971). (2) J. Close and P. A. Nielson, Resistance of a strain of Pseudomonas cepacia to esters of p-Hydroxybenzoic acid, Applied & Environmental Microbiology, 31, 718-722 (1976). (3) B. T. Decicco, E. C. Lee, and J. V. Sorrentino, Factors affecting survival of Pseudomonas cepacia in decongestant nasal sprays containing thimerosal as preservative, Journal of Pharmaceutical Sciences, 71, 1231-1234 (1982). (4) H. Dixon and C. S. Dolan, Occurrence of mercury-resistant Pseudomonas in emulsion formulations preserved with phenylmercuric acetate, InternationalJ. of Cos. Science, 3, 261-266 (1981). (5) R. L. Anderson, R. L. Berkelman, et al., Investigations into the survival of Pseudomonas aeruginosa in poloxamer-iodine, Applied and Environmental Microbiology, 47, 757-762 (1984). (6) D. S. Orth and C. M. Lutes, Adaptation of bacteria to cosmetic preservatives. Cosmetics & Toiletries, 100, 57-64 (February, 1985).