Journal of
`Applied Bacteriology
`
`Edited by
`
`D.E. Stewart-Tull, G.I. Barrow
`
`and R.G. Board
`
`Volume 72, 1992
`
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`Copyright © 1992 by The Society for Applied Bacteriology
`ALL RIGHTS RESERVED
`
`Nopart of this volume may be reproducedin any form, 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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`Kinetic evaluation of claimed synergistic paraben
`combinations using a factorial design
`
`D. Gilliland, A. Li Wan Po andE. Scott
`The Drug Delivery Research Group, The School of Pharmacy, The Queen's University of Belfast, Northern Ireland
`3738/07/91: accepted 20 September 1991
`D. GILLILAND, A. LI WAN PO AND E. SCOTT. 1992. The antimicrobial effects of methyl and
`propyl parabensare investigated, with Escherichia coli as test organism, with a view to
`determining whether the parabensact synergistically. At appropriate concentrations, the
`parabenskilled E. colt cells accordingto first order kinetics and the bactericidal effects were
`quantified by thefirst order kill rate constants. Combinations of methyl or propyl parabens,
`at concentrations which slow downorinhibit bacterial growth whenusedsingly, produced
`definite kill, In this sense, the parabens are therefore synergistic since in combination they
`produce aneffect which is not observed when they are used singly. This effect is not true
`synergism as shown bytheresults of our experiments with a factorial design. Analysis of
`variance indicated no significant interaction between the two parabens.
`
`INTRODUCTION
`Combinationsof 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 ofthe 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 parabensact additively or syner-
`gistically. A kinetic method and a factorial experimental
`design were used.
`
`MATERIALS AND METHODS
`
`Preparation of media
`(g/l):
`The
`chemically
`defined medium contained
`Na,HPO,, 11-45; KH2PO,, 1:-4025; (NH,4)2SO,4, 1-87;
`M,SO,,
`0-187;
`D-glucose,
`0-909;
`CaCl,(2H,0),
`
`Correspondence to: Prof. A. Li Wan Po, The Drug Delivery Research
`Group, The School ofPharmacy, The Queen's University of Belfast, 97
`Lisburn Road, Belfast BT9, Northern Treland.
`
`5 x 10-7. The pH was
`1:245 x 1075; FeSO, (7H,0),
`adjusted to 6-9 with dilute HCl. All chemicals were of ana-
`lytical reagent quality.
`
`Preparation of inoculum
`Escherichia coli NCIB 8545 was maintained on Tryptone
`Soya Agar (Oxoid)slants at 4°C. A loopful of the organism
`was added to 100 mlof 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 1 x 10® cfu/ml. An inoculum of 1 x 10°
`cfu/ml was prepared by filtering the culture (100 ml),
`under aseptic conditions,
`through a 0-45 ym membrane
`filter and washing with 100 mloffresh, pre-warmed media.
`The organisms were then resuspended in 10 ml of media to
`give the final inoculum.
`
`Preparation of test solutions
`The appropriate weights of the methyl and propyl esters of
`p-hydroxybenzoic acid (Sigma) were added to 1
`1 of
`medium and placed in a sonic bath for up to 4h 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 ina
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`Measurementof microbial numbers
`
`After inoculation of the test solutions viable counts of
`microbial numbers were madeat regular intervals. A 1 ml
`sample was
`removed from the test solution and serial
`10-fold dilutions were made in 0-1% 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.
`
`Statistical analysis
`
`A 2? factorial design was used in our study. The low
`paraben levels corresponded to 0:12% w/v for methyl
`paraben and 0-012% w/v for propyl paraben. The high
`concentrations corresponded to 0:14% w/v for methyl
`paraben and 0-014% 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
`0-12% w/v and 0-:014% w/v concentrations, respectively,
`produced definite kill of the E. colt cells (Fig. 1). There was
`no obvious lag phase in the kill curve and the data closely
`fitted first order kinetics. Table | 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 0-12 and
`
`Lncfu/ml
`
` 100
`
`200
`Time (min)
`
`300
`
`400
`
`Fig. 1 A semi-logarithmic plot of the effect of 0-12% methyl
`paraben and 0-14%propyl paraben, in combination, on
`Escherichia coli. (Error bars are s.£., 1 = 5.)
`
`Table 1 The effect of concentration of methyl and propyl
`parabensalone and in combination on Escherichia coli
`
`Concentration
`(% w/v)
`
`Control
`
`0-012 propyl paraben
`0-014 propyl paraben
`0-12 methyl paraben
`+0-012 propyl paraben
`0-12 methyl paraben
`+ 0-014 propyl paraben
`0-14 methyl paraben
`+0-012 propyl paraben
`0-14 methyl paraben
`+0-014 propyl paraben
`
`First order rate
`constant (/min)
`
`0:0143
`
`0-0105
`0-0110
`
`—0-0091
`
`—0-0289
`
`—0-0385
`
`— 0-0649
`
`S.E.
`n= 20
`
`0-0003
`
`0-0003
`0-0002
`
`0-0005
`
`0-0032
`
`0-0034
`
`0-0026
`
`0:14% w/v for methyl paraben and 0-012 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 effect with the two parabensat the
`concentrations used (Table 2).
`In a factorial experiment
`such as the present one,interaction is shown by a changein
`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 &
`Cox 1957) as was demonstrated in this study when the
`methyl paraben concentration was increased while main-
`taining the propyl paraben concentration at either 0-012%
`w/v (low) or 0-014% 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 moreanti-
`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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`Sres
`Table 2 Analysis of variance for the rate
`Seq SS Adj SS Adj MS FDF PeALeetekeo
`constants from first orderkill curves of
`Methyl paraben
`methyl and propy! parabens against
`Escherichia colt
`concentration
`Propyl paraben
`0-010512
`concentration
`0-000914
`Combination
`0-010632
`76
`Error
`0-042654
`79
`Total
`ogiaie
`DF,Degrees of freedom; Seq SS, uncorrected sum of squares; Adj SS, adjusted sum of
`squares; Adj MS,adjusted mean squares; F,F-ratio.
`
`1
`
`1
`
`0-21316
`
`0-021316
`
`0-021316
`
`152-37
`
`<0-001
`
`0-010512
`0-000914
`0-010632
`
`0-010512
`0-000914
`0-000140
`
`75-14
`1:39
`
`<0-001
`0-243
`
`:
`
`a
`
`400
`
`ba
`t
`
`=Ss
`& 2e+7
`;
`2ooO
`&2
`
`9>
`
`le+7
`
`a
`
`Low
`0-12
`
`High
`0:14
`
`a
`
`5
`
`ua
`
`a
`
`Oe+0O
`
`0
`
`100
`
`200
`Time (min)
`
`Oo
`
`300
`
`0-07
`
`0-06
`
`0-05
`
`0-04
`
`0-03
`
`0-02
`
`0-01
`
`0-00
`
`
`
`
`
`Rateconstant(/min)
`
`Methyl! paraben concentration (% w/v)
`
`Fig. 2 Results of factorial design experimentto evaluate the effect
`of methyl and propyl paraben combinations on thekill rate
`constantof Escherichia coli. (Error bars are s.£., » = 20.) , 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 et a/. 1990). With this approach we
`were not able to show any synergism in the concentration
`ranges 0:12% w/v and 0-14% w/v for methyl paraben and
`0-012% w/v and 0-014% w/v for propyl paraben.
`It
`is interesting to note however that 0:12% w/v and
`0:14% w/v methyl paraben when used alone produced a
`bacteriostatic effect (Fig. 3). Propyl paraben at 0-012% w/v
`
`Fig. 3 The survival of exponential phase Escherichia coli cells in
`chemically defined media in the presence of 0:12%w/v and
`0-14%w/v methyl paraben.(Error barsare s.£., 2 = 5.) D,
`0-12% methyl paraben; @, 0:14% methyl paraben
`
`and 0-014% 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 dose-response relationship is not linear but sigmoidal
`(Goldstein et
`af.
`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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`7e+8
`
`6e+8
`
`5e+8
`
`4e+8
`
`se+8
`
`2e+8
`
`le+8
`
`
`
`
`
`Viablecount(cfu/ml)
`
`
`
`Lnviablecount
`
`a
`
`o
`
`100
`
`
`Ge +02eeEeeeee
`0
`100
`200
`300
`400
`Time (min)
`
`
`
`300
`200
`Time (min)
`
`400
`
`500
`
`Fig. 4 The growth of exponential phase Escherichia coh cells in
`chemically defined media in the presence of 0-012% propyl
`paraben andin the presence of 0-014% propyl paraben. (Error
`bars are S.E., 2 = 5.) 1), 0-012%propyl paraben; @, 0:014%
`propyl paraben
`
`REFERENCES
`
`BERENBAUM, M.C. (1977) Synergy, additivism and antagonism
`in immunosuppression. A critical review. Clinical and Experi-
`mental Immunology 28, 1-18.
`Cocuran, W.G. & Cox, G.M. (1957) Factorial experiments.
`In Experimental Designs, pp. 148-181. New York: Wiley.
`GoLpsTEIN, A., ARoNow, L. & KALMAN, S.M. (1974)
`Molecular mechanisms of drug action. In Principles of Drug
`Action, 2nd edn. Ch. 1, pp. 1-128. New York: Wiley Interna-
`tional.
`
`Fig. 5 The survival of exponential phase Escherichia colt cells in
`chemically defined media in the presence of combinations of
`methyl and propyl parabensat high (H) and low (L)
`concentrations. (Error bars are s.£.,2 = 5.) 1], L methyl + L
`propyl; @, H methyl + L propyl; Mf, L methyl + H propyl; O,
`H methyl + H propyl
`
`MatTHews, J.N.S., ALTMAN, D.G., CAMPBELL, M.J.
`& Royston, P.
`(1990) Analysis of serial measurements in
`medical research. British Medical Journal 300, 230-235.
`MOELLERING, R.C.
`(1979) Antimicrobial
`synergism—an
`elusive concept. Journal of Infectious Diseases 140, 639-641.
`Norben, C.W. (1982) Problems in determination of antibiotic
`synergism in vitro. Reviews ofInfectious Diseases 4, 276-281.
`
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