de Solis, Nilka M.G.; (1993) Effect of plasmids that confer preservative-resistance on the performance of bacteria in preservative efficacy tests. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The effect of plasmids on the efficacy of pharmaceutical preservatives in simple aqueous preserved systems was assessed. This was done by determining minimum inhibitory concentrations (MICs), susceptibilities of strains to bactericidal concentrations of preservatives, and susceptibilities of strains to preservative challenge using the conditions of the Preservative Challenge Test of the British Pharmacopoeia (1988). The pharmaceutical preservatives used were: benzalkonium chloride (BZC), cetrimide (CTAB), propamidine isethionate (PI), dibromopropamidine isethionate (DBPI), chlorhexidine gluconate (CG) and phenylmercuric nitrate (PMN). MIC data for all the preservatives tested showed the plasmid-less Pseudomonas aeruginosa strain NCIB 8626 to be overall the most resistant organism. This strain recorded MICs (in pg/ml) to BZC = 150, CTAB = 300, PI = 150, DBPI = 75, CG = 5.0 and PMN = 30. However, the plasmid-containing strains S. aureus SA1325 (pSK l) and E. coli 343/113 (R471-1) paralleled or surpassed the resistance levels of the pseudomonad for certain of the preservatives. MICs for the staphylococcus of 150 pg/ml and for the E. coli of 40 pg/ml were recorded against PI and PMN, respectively. Short-term killing rate experiments compared the sensitivities of plasmid-containing and plasmid-less strains to bactericidal concentration of the preservatives. In contrast to the MIC data, bactericidal activity experiments showed P. aeruginosa to be the most sensitive organism to 5 pg/ml CG, 1 and 10 pg/ml PMN, 5 mg/ml PI and 1 mg/ml DBPI. Survival of S. aureus strain SA1325 (pSKl) in 5 pg/ml CG was 100-fold greater after 90 min when compared to its isogenic parent SA 1439. Results of the bactericidal experiments that correlated with MIC data were obtained for the pseudomonad in 10 3 pg/ml BZC and in 10 pg/ml CTAB solution in which the organism was highly resistant. However, in most cases the resistance levels of the plasmid-containing S. aureus and E. coli strains surpassed the resistance levels exhibited by the pseudomonad. It is concluded this was due to the method of preparation of inocula, which may have sensitized the pseudomonads to some of the preservatives. The efficacies of three different aqueous concentrations of BZC (10, 30 and 100 pg/ml), CTAB (10, 30 and 100 pg/ml), PMN (1, 10 and 20 pg/ml) and CG (5, 20 and 100 pg/ml) were measured using the protocol of the BP Preservative Challenge Test. These experiments were performed to determine if the presence of plasmids in test strains made any difference to the compliance of preservative systems with the test. The results demonstrated increased survival of S. aureus SA1325 (pSK l) in 10 pg/ml BZC or CTAB and in 1 pg/ml PMN when compared to the plasmid-less S. aureus strains. The pseudomonad produced a pattern of death and regrowth in 10 pg/ml CTAB that was reproducible. It was concluded that plasmids do provide bacteria with the necessary protection to grow, as for example in MIC experiments, or survive, as in bactericidal tests or BP challenge tests, in unfavourable environments. However, the in-use preservative concentrations tested still provided an acceptable degree of protection against plasmidcontaining and persistent organisms.

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