Journal of
`
`Applied Bacteriology
`
`Edited by
`
`D.E. Stewart-Tull. G.l. Barrow
`
`and HG. Board
`
`Volume 72, 1992
`
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`Copyright © 1992 by The Society for Applied Bacteriology
`ALL RIGHTS RESERVED
`
`No part of this volume may be reproduced in any fun-n, by
`photostat, microfilm, or any other means, without written
`permission from the Society
`
`ISSN 0021-8847
`
`Published by
`Blackwell Scientific Publications Ltd
`OXFORD LONDON EDINBURGH BOSTON
`MELBOURNE
`PARIS
`BERLIN
`VIENNA
`
`Printed in Great Britain
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`Journal of Applied Bacteriology 1992, 72, 258—261
`
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`
`Kinetic evaluation of claimed synergistic paraben
`combinations using a factorial design
`
`D. Gilliland, A. Li Wan Po and E. Scott
`The Drug Delivery Research Group, The School of Pharmacy. The Queen '5 University of Belfast, Northern ireland
`
`3738,’07/91 : accepted 20 September 1991
`
`D. GlLLILAND, A. LI WAN PO AND E. SCOTT. 1992. The antimicrobial effects of methyl and
`propyl parabens are investigated, with Escherichia rah as test organism, with a view to
`determining whether the parabens act synergistically. At appropriate concentrations, the
`parabens killed E. 6011' cells according to first order kinetics and the bactericidal effects were
`quantified by the first order kill rate constants. Combinations of methyl or propyl parabens,
`at concentrations which slow down or inhibit bacterial growth when used singly, produced
`definite kill. In this sense, the parabens are therefore synergistic since in combination they
`produce an effect which is not observed when they are used singly. This effect is not true
`synergism as shown by the results of our experiments with a factorial design. Analysis of
`variance indicated no significant interaction between the two parabens.
`
`INTRODUCTION
`
`Combinations of antimicrobial agents are widely used both
`for
`treating diseases and for preserving pharmaceutical
`systems. The rationale is that by using combinations, the
`activity spectrum may be broadened and the agents
`involved may act additively or synergistically. Sometimes
`one of the agents in a combination may by itself be inactive.
`Despite the widespread use of antimicrobial combinations,
`clinical evidence for synergy is difficult to generate even
`with in vitro systems. The best method for demonstrating
`synergism is the subject of controversy (Berenbaum 1977).
`A widely used combination preservative system for which
`there is much in-use evidence for at least an additive anti—
`microbial effect
`is
`the methyl paraben/propyl paraben
`mixture.
`In this report we describe studies designed to
`evaluate whether the two parabens act additively or syner—
`gistically. A kinetic method and a factorial experimental
`design were used.
`
`5 x 10—7. The pH was
`1-245 X 1075; FeSO4 (7H20),
`adjusted to 6-9 with dilute HCI. All chemicals were of ana-
`lytical reagent quality.
`
`Preparation of Inoculum
`
`Escherichia wh‘ NCIB 8545 was maintained on Tryptone
`Soya Agar (Oxoid) slants at 4°C. A loopful of the organism
`was added to 100 ml of sterile media and grown at 37“C
`overnight in a shaking waterbath at 100 rev/min. Transfers
`of organism were made daily for 2 d. On the third transfer
`the organisms were allowed to grow to an optical density
`reading of 0-1 at 540 nm (Corning colorimeter 254). This
`provided cells in the exponential phase of growth. The
`absorbance value of 0-1 at 540 nm was found to be approx-
`imately equal to l x 10E cfu/rnl. An inoculum of 1 x 109
`cfu/ml was prepared by filtering the culture (100 ml),
`under aseptic conditions,
`through a 0-45 pm membrane
`filter and washing with 100 ml of fresh, pre-warmed media.
`The organisms were then resuspended in 10 ml of media to
`give the final inoculum.
`
`MATERIALS AND METHODS
`
`Preparatlon of test solutions
`
`Preparation 01 media
`
`(g/l):
`defined medium contained
`chemically
`The
`Nazi-1P0,“ 11-45; KHZPO4, 1-4025; (N144); 304, 1-87;
`M5804,
`0-187;
`o—glucose,
`0-909;
`CaC12(2H20),
`
`Corrupmdenrt- to : Prof: A. Li Wm: Pa, The Drug Delft-try RESt’fln’h
`Group, The Srhoa! ofPharmary, The Queen's University affietflfint, 97
`Lisbunt Road, Belfast 37“), Northern Ireland.
`
`The appropriate weights of the methyl and propyl esters of
`p-hydroxybenzoic acid (Sigma) were added to 1
`l of
`medium and placed in a sonic bath for up to 4 h to aid
`solubilization. The solution was filter-sterilized and 100 ml
`of test solution dispensed into 250 ml
`flasks. Medium
`without any parabens was employed as control. Before
`inoculation, the test solutions were maintained at 37°C in a
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`ANTIMICROBIAL PARABEN COMBINATIONS 259
`
`Measurement of mlcroblal numbers
`
`After inoculation of the test solutions viable counts of
`
`microbial numbers were made at regular intervals. A 1 ml
`sample was
`removed from the test solution and serial
`10—fold dilutions were made in 01% peptone water. One
`ml volumes of the dilutions were plated by the pour plate
`method with Isosensitest Agar (Oxoid). After incubation at
`37°C for 20 h, colonies were counted and the number of
`cfu/ml evaluated.
`
`Statistleal analyels
`
`A 22 factorial design was used in our study. The low
`paraben levels corresponded to 012% w/v for methyl
`paraben and 0012% w/v for propyl paraben. The high
`concentrations corresponded to 014% w/v for methyl
`paraben and 0014"o w/v for propyl paraben. In total 80
`kinetic runs were performed over 16 d and each day
`involved five kinetic runs. One kinetic run consisted of an
`
`enumeration of the number of colony-forming cells at pre-
`determined time intervals. Within—day variability was of the
`same order as between—day variability. Therefore blocking
`was not required.
`
`RESULTS
`
`Combination of methyl paraben and propyl paraben at
`012% w/v and 0014% w/v concentrations, respectively,
`produced definite kill of the E. rah cells (Fig. 1). There was
`no obvious lag phase in the kill curve and the data closely
`fitted first order kinetics. Table 1 lists the appropriate sta—
`tistics for the rate constants at all combinations studied.
`
`In order to investigate whether the two parabens in com-
`bination produced a synergistic effect we chose 012 and
`
`
`
`200
`TII'HE [mml
`
`300
`
`400
`
`Lncfu/ml
`
`0
`
`I00
`
`Fly. 1 A semi—logarithmic plot of the efl’eet of 012% methyl
`paraben and 014% propyl paraben, in combination, on
`Escherichia 5011'. (Error bars are 5.15., n I 5.)
`
`Table 1 The effect of concentration of methyl and propyl
`parabens alone and in combination on Escherichia 6011'
`
`Concentration
`
`(”/9 w/v)
`
`Control
`
`0-012 propyl paraben
`0-014 propyl paraben
`012 methyl paraben
`+0012 propyl paraben
`012 methyl paraben
`+0014 propyl paraben
`014 methyl paraben
`+0012 propyl paraben
`014 methyl paraben
`+0014 propyl paraben
`
`First order rate
`
`constant (fmin)
`
`0014:!
`
`00105
`00110
`
`—0-0091
`
`—00289
`
`-—00385
`
`—0-0649
`
`5.12.
`
`n = 20
`
`0-0003
`
`00003
`0-0002
`
`00005
`
`00032
`
`00034
`
`00026
`
`014% w/v for methyl paraben and 0012 and 0-014% w/v
`for propyl paraben because pilot studies showed that at
`lower concentrations the combinations were often only bac—
`teriostatic and consistent kill rate constants could not be
`
`calculated. Combinations of higher paraben concentrations
`on the other hand killed the bacteria too quickly for the
`required sampling to be carried out satisfactorily. Conse-
`quently, the kill rate constants derived from the kill curves
`were again imprecise.
`the within—day
`Evaluation of the results showed that
`variability was of the same order as the between—day varia-
`bility. Blocking was therefore not advantageous and each
`experimental kill curve was considered to be an indepen-
`dent run.
`
`Analysis of variance of the rate constants showed that
`there was no synergistic efi'ect with the two parabens at the
`concentrations used (Table 2).
`In a factorial experiment
`such as the present one, interaction is shown by a change in
`slope of the kill rate constant and concentation plot when
`the concentration of the interactant is changed. Absence of
`interaction is conversely shown by parallel lines (Cochran 8t
`Cox 1957) as was demonstrated in this study when the
`methyl paraben concentration was increased while main-
`taining the propyl paraben concentration at either 0012%
`w/v (low) or 0014% w/v (high) (Fig. 2) and, correspond-
`ingly, when the propyl paraben concentration was increased
`while maintaining the methyl paraben concentration con—
`stant.
`
`concept
`elusive
`an
`synergism is
`Antimicrobial
`(Moellering 1979). Although the most widely accepted defi—
`nition of this term is ‘the joint action of two or more anti—
`microbial agents to produce an effect which is greater than
`the sum of the individual effects when the drugs are used
`alone’,
`the most appropriate methodology used to prove
`synergy is still controversial. Norden (1982) showed that
`discordant conclusions are reached when results from the
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`260 D. GlLLILAND ET AL.
`E
`
`n
`.
`_——————F—_
`Table 2 Analysts of variance for the rate
`DF
`Seq SS
`Adj SS
`Adj MS
`F
`P
`EH constants from first order kill curves of
`Methyl paraben
`concentration
`Propyl paraben
`0010512
`1
`concentration
`0000914
`1
`Combination
`0010632
`76
`Error
`0042654
`79
`Total
`—-*.__—______
`
`1
`
`021316
`
`0021316
`
`0021316
`
`152-37
`
`< 0001
`
`methyl and propyl parabens against
`Escherichia calf
`
`0010512
`0000914
`0010632
`
`0010512
`0000914
`0-000140
`
`75- 14
`1-39
`
`(0001
`0243
`
`DF, Degrees of freedom; Seq SS, uncorrected sum of squares; Adj SS, adiusted sum of
`squares; Adj MS, adjusted mean squares; F, F—ratio.
`
`l
`
`i
`
`1
`
`{
`
`i
`
`”E:5
`g 2e+T
`DOU
`E
`2.G
`
`g>
`
`19+?
`
`Oe+O
`
`n
`
`E
`
`11
`
`1::
`
`n
`
`H
`
`O
`
`100
`
`200
`Tune (min)
`
`300
`
`400
`
`Fly. 3 The survival of exponential phase Escherichia coli cells in
`chemically defined media in the presence of 012"" w/v and
`014% w/v methyl paraben. (Error bars are 5.5., n = 5.) [:l,
`012% methyl paraben; ., 014% methyl paraben
`
`and 0014% w/v on the other hand allowed bacterial
`
`growth to continue when used singly (Fig. 4). Yet every
`combination within those concentration ranges produced
`bacterial kill (Fig. 5). The results therefore clearly show
`that in this sense the combination is synergistic, that is the
`combination produces an effect (observable kill) which is
`not seen when the agents are used singly.
`To explain why there is this apparent sudden change
`from a bacteriostatic to a bactericidal effect for the paraben
`combination it is worth noting that with most drug entities,
`the dosekresponse relationship is not linear but sigmoidal
`(Goldstein et
`at.
`1974). Therefore apparently abrupt
`changes in potency are often observed. The danger of mis-
`taking this change for synergism has been highlighted by
`Berenbaum (1977).
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`
`
`
`RoleconstantUrnin)
`
`0-07
`
`0-06
`
`005
`
`C) 9
`
`0-03
`
`0-02
`
`O-OI
`
`0-00
`
`a
`
`Law
`0-|2
`
`1
`
`High
`0-14
`
`Methyl poroben concentration (% w/v)
`
`Fig. 2 Results of factorial design experiment to evaluate the effect
`of methyl and propyl paraben combinations on the kill rate
`constant of Escherichia coli. (Error bars are S.E., n : 20.) E], Low
`propyl paraben; ., high propyl paraben
`
`checker board method are compared with those from kill
`curves that used two time points. This is not surprising
`since the checker board method uses the minimum inhibi-
`tory concentration while the kill curve method uses rate of
`
`kill as end-points. In our present method we have used the
`rate constants as end-points since this summary statistic
`enables us to avoid multiple comparisons of serially corre-
`lated data (Matthews er a1. 1990). With this approach we
`were not able to show any synergism in the concentration
`ranges 012% w/v and 014% w/v for methyl paraben and
`0012% w/v and 0014% w/v for propyl paraben.
`[t
`is interesting to note however that 012% w/v and
`014% w/v methyl paraben when used alone produced a
`bacteriostatic effect (Fig. 3). Propyl paraben at 0-012% w/v
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`ANTIMICROBIAL PARABEN COMBINATIONS 261
`
`I
`
`Te+8
`
`63 +8
`
`5e+8
`
`4e+8
`
`Be +8
`
`2e +8
`
`|e+8
`
`
`
`
`
`Viablecount(Clo/mil
`
`/
`
`
`02 +0 _—-I—%J___l
`O
`IOO
`200
`300
`400
`Time (min)
`
`Fla. 4 The growth of exponential phase Escherichia coli cells in
`chemically defined media in the presence of 0-012% propyl
`paraben and in the presence of ”014% propyl paraben. (Error
`bars are 5.1-”... n = 5.) D, 0-012% propyl paraben; ., 0-014%
`propyl paraben
`
`REFERENCES
`
`BERENBAUM, M.C. (I977) Synergy, additivism and antagonism
`in immunosuppression. A critical review. Clinical and Experi-
`mental Immunology 28, 1—18.
`COCHRAN, W.G. 8: Cox, G.M. (1957) Factorial experiments.
`In Experimemm' Designs, pp. 1487181. New York: Wiley.
`(1974)
`GOLDSTEIN, A., ARONow. L. &: KAI.MAN, S.M.
`Molecular mechanisms of drug action. In Principles of Drug
`Action, 2nd edn. Ch. I, pp. 1—128. New York: Wiley Interna-
`tional.
`
`
`
`Lnviablecount
`
`
`
`O
`
`IOO
`
`300
`200
`Time (min)
`
`400
`
`500
`
`Fig. 5 The survival of exponential phase Escherichia rali cells in
`chemically defined media in the presence of combinations of
`methyl and propyl parabens at high (H) and low (L)
`concentrations. (Error bars are s.£., n = 5.) E], L methyl + L
`propyl; ., H methyl + L propyl; I, L methyl + H propyl; O,
`H methyl + H propyl
`
`MATTHEWS, j.N.S., ALTMAN, D.G., CAMPBELL, MJ.
`& ROYSTON, P.
`(1990) Analysis of serial measurements in
`medical research. British Medical Journal 300, 230435.
`MOELLERING, R.C.
`(1979) Antimicrobial
`synergismfln
`elusive concept. Journal aflnfectious Diseases 140, 639—64].
`NORDEN, C.W. (1982) Problems in determination of antibiotic
`synergism in virro. Reviews ofInfectious Diseases 4, 276—28].
`
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