420 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 8 7 6 5 4 '• I I I I 0 1 2 ,3 7 Days Figure 6. Adaptation of P, aeruginosa P5 to 2.0% imidazolidinyl urea after pre-incubation in dilute nu- trient broth (V•), dilute broth containing 1% irnidazolidinyl urea (+), and dilute broth containing 1% imidazolidinyl urea and 105• emulsion (0). (Figure 6). The population pre-incubated in unpreserved broth decreased rapidly for 48 hours after inoculation into 100% product. A similar adaptation pattern was noted for strain 495, RC, P6, and 445. The ATCC strain could not be adapted to the preserva- tive. The cells died off rapidly, even after pre-incubation in low levels of imidazolidinyl urea. A similar adaptation phenomenon may occur in the manufacturing environment on equipment which is not thoroughly cleaned. Organisms may adapt to dilute product left on the equipment and enter an exponential growth phase when inoculated into a subsequent production batch. Growth patterns in the emulsion preserved with imidazolidinyl urea and methyl par- aben were measured because this dual system is frequently employed in cosmetic products (6, 10). Most of the product isolates did not demonstrate resistance to the imidazolidinyl urea, methyl paraben preservative system. Only strain 445 was resistant to the combination of imidazolidinyl urea (0.5%) and methyl paraben (0.3%) (Figure 7). The resistant cell population decreased for the first 48 hours from 104 cfu/ml down to less than 10 cfu/ml, but increased to 105 cfu/ml by day 7. This strain was inhibited at higher methyl paraben concentrations. All other product isolates died within 24 hours after inoculation into the products containing 0.3% methyl paraben. Additional work is being carried out to further study strain 445 and its mechanism of resistance to methyl paraben. SPECTROPHOTOMETRIC ASSAY FOR IMIDAZOLIDINYL UREA To elucidate the mechanism of imidazolidinyl urea resistance in these organisms, the

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