4/ ".1 j
`
`-- 5‘
`
`Journal of
`
`Applied Bacteriology
`
`Edited by
`
`D.E. Stewart—Tull, GA. Barrow
`
`and FLG. Board
`
`Voiurne 72,1992
`
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`

`Copyright © I992 by The Society for Applicd Bacteriology
`ALL RIGHTS RESERVED
`
`No part of this volume may be reproduced in any form, by
`photostat, microfilm, or any other mans, 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, 12, 252—25?
`
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`
`
`The bactericidal activity of a methyl and propyl parabens
`combination: isothermal and non-isothermal studies
`
`D. Gilliland, A. Ll Wan Po and E. Scott
`The Drug Delivery Research Group, The School of Pharmacy. The Queen ’5 University of Belfast. Northern ireiand
`
`3739/07/91: accepted 20 September 1991
`
`D. GILLILAND. A. Ll WAN PO AND E. SCOTT. 1992. The effect of temperature on the kill rate of
`
`Escherichia coli by methyl and propyl parabens was studied. The kill kinetics was first order.
`It was shown that the Arrhenius equation provided a good model for describing the
`
`relationship between the first order rate constant and the temperature. The activation energy
`
`was found to be 274 kJ/rnol for exponential phase cells and 168 kjlmol for stationary phase
`cells. Exponential phase cells were much more susceptible to the lethal effects of the parabens
`than were the stationary phase cells. For example, at 34°C stationary phase cells, in
`
`chemically defined media, had a kill rate constant of 0-072/h while the corresponding value
`
`for exponential phase cells was 0-238/h. In water the rate of kill for exponential phase cells
`was even faster giving a rate constant of 5-25/h at 34°C. Non-isothermal kinetic testing was
`not found to be useful for modelling bacterial kill kinetics because we could not achieve the
`
`precision required in bacterial enumeration.
`
`INTRODUCTION
`
`In the presence of a bactericidal antimicrobial agent the
`rate of kill of microbial cells generally increases as the tem-
`perature increases (Lynn & Hugo 1983). The efl'ect of tem-
`perature is often expressed in terms of a temperature
`coefficient (Pflug 1972), usually measured as the change in
`rate constant over a 10"C increase in temperature and
`referred to as the Q10. Those compounds with high tem-
`perature coefficients exhibit greater increases in activity
`with increasing temperature. However,
`the temperature
`coefficient value tends to vary over the temperature range
`studied, with decreasing values at higher
`temperature
`ranges (Karabit e! 41. 1985). The Q10 values also vary from
`one organism to another for the same antimicrobial agent
`(Karabit e: a]. 1986).
`Both isothermal and non-isothermal methods were used
`
`to ascertain the effect of temperature on bacterial kill rates.
`
`THEORY
`
`Under standardized conditions bacterial death is often
`
`exponential as described by eqn. (1)
`
`N=N0e”‘-'
`
`(1)
`
`Correspondence to: Prof A. Li Wan Pa, The Drug Delivery Research
`Group, The School ofPhormary, The Queen's University offlelfarr, 97
`Lisbon: Road, Belfast 37“), Northern Ireland.
`
`where N is the number of organisms surviving at time t,
`No is the initial number of organisms and kl
`is the rate
`constant describing the kill.
`One of the major factors affecting the kill rate of bacteria
`is the temperature. To describe the effect of temperature
`many authors have used the Arrhenius equation (eqn. (2))
`
`k = Ae“E'“m
`
`(2)
`
`where k is the rate constant, R is the gas constant, T is the
`temperature in degrees Kelvin, A is the pre—exponential
`constant and E, is the activation energy.
`To test the validity of the Arrhenius equation, adherence
`of the kill curve to an appropriate rate equation is first
`established. Most commonly,
`the first order model (eqn.
`(1)) is appropriate. If validated, experimental verification of
`the Arrhenius equation then involves calculation of appro-
`priate rate constants (k) at a number of different
`tem-
`peratures (T) and their statistical evaluation. Generally, the
`linear form of eqn. (2) is used and adherence to the model
`is shown by a linear relationship between log|= (k) and 1/ T.
`Many authors have shown that when the range of tem-
`perature is wide, marked deviations from linearity are
`observed when loge(k) and l/T are plotted. Models put
`forward
`to
`represent
`the
`rate
`constant—temperature
`relationship in such cases have included the square root
`model (Ratkowsky at a]. 1983) and the Schoolfield model
`(Schoolfield st e1. 1981) represented by eqns. (3) and (4-),
`respectively.
`
`fl = bu“ — Tani] — sure-01}
`
`(3)
`
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`BACTERICIDAL EFFECT OF PARABENS 253
`
`where T is the temperature in degrees Kelvin, Tmin is the
`theoretical minimum temperature for growth, Tm“ is the
`theoretical maximum temperature for growth, b and c are
`constants and k is the rate constant.
`
`logck = A + B/T— logeT
`
`+ loge [l + C(“Dm + elGJ'Hm]
`
`(4)
`
`where A, B, D, F, G and H are constants, k is the rate
`constant and T is the temperature in degrees Kelvin.
`Data for testing temperature—kill rate relationships are
`collected as described above (eqns (2H4)) and each kill
`curve is
`recorded at a constant
`temperature. Such an
`experimental set-up is referred to as isothermal testing. An
`approach which has gained some measure of acceptance in
`the stability testing of pharmaceutical products is that of
`non—isothermal testing. In this case temperature is varied
`continuously during the experiment so that,
`theoretically,
`temperature effects on kill rates can be derived from one
`single experiment. The logic behind the method is as
`follows.
`
`Suppose that from an experiment, carried out at constant
`temperature, the kill curve adheres to eqn (1). A plot of
`loge (N) or log, (N/No) against time will be linear. If we
`now increase the temperature during the experimental run,
`the line will usually curve down as shown in Figs. 1a and
`lb. Starting the experiment below the optimum tem-
`perature for growth and increasing the temperature during
`the experiment will slow down the observed kill rate if the
`temperature effect on growth rate exceeds that on the
`preservative-induced kill rate. The two effects will counter—
`act each other.
`In that case the kill plot will show an
`upward curve.
`
`A curve such as that shown in Fig. 1b can usually be
`satisfactorily modelled by a low order polynomial equation
`which can be written as
`
`logc(N)=f(t)=flo+alt+a2;2+...
`
`(S)
`
`The derivative of this equation gives the rate constant k at
`the prevailing temperature T at time t
`
`dDOge (ND/df=k1=al+2azt+...
`
`(6)
`
`Therefore, provided we have the temperature at time t,
`the corresponding rate constant can be calculated. The
`
`appropriate calculations can be easily done using both stan-
`dard statistical computer packages and non-isothermal spe-
`cific programs (Li Wan Po et a1. 1983). More detailed
`descriptions of non-isothermal stability testing methodology
`are given elsewhere (Hempenstall er a1. 1983).
`
`MATERIALS AND METHODS
`
`Preparation of medla
`
`(g/l):
`defined medium contained
`chemically
`The
`NazHPO4, 11-45; KH2P04, 1-4025; (NH4)ZSO4, 1'87;
`MgSO4,
`0'187; D-glucose,
`0-909; 0102
`(21-120),
`1-245 x 10—5; FeSO4(7H20),
`5 XII)”. The pH was
`adjusted to 6‘9 with dilute HCl. All chemicals were of ana-
`lytical reagent quality.
`
`Preparation of the lnoculum at exponentlal phase
`cells
`
`Escherichia ml! NCIB 8545 was maintained on Tryptone
`Soya Agar (Oxoid) slopes at 4°C. A loopful of the organism
`was added to 100 ml of sterile media and grown overnight
`at 370C 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 01 at 540 nm (Corning calorimeter 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 10B cfu/ml. An inoculum of l X 109
`cfu/ml was prepared by filtering the culture (100 ml),
`under aseptic conditions, through a 0-4-5 ,um membrane 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.
`
`Preparallon of the inoculum of statlonary phase cells
`
`The same procedure was carried out for the preparation of
`exponential phase cells except that instead of preparing a
`third transfer the overnight cells were used to prepare the
`
`Fig. 1 Theoretical first order kill curve for
`micro-organisms at (a) constant
`temperature and (b) when the temperature
`is increased continuously throughout the
`experiment
`
`(0)
`
`Lncfu/ml—-—b
`
`lb)
`
`Hfib
`Lncfu/ml
`
`Time —’
`
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`

`254 D. GILLILAND ET AL.
`
`
`inoculum. The culture was filtered, under aseptic condi-
`tions, through a 0-45 pm membrane filter and washed with
`100 ml of pre—warmed media. The organisms were then
`resuspended in 10 m1 of media and the density adjusted so
`that a l
`: 10 dilution gave an absorbance reading, at 540
`nm, of approximately 0-1.
`
`Preparatlon 01 test solutions
`
`The appropriate weight of the methyl and propyl esters of
`p—hydroxybenzoic acid (Sigma) were added to 1
`l of
`medium, or water, and placed in a sonic bath for up to 4 h
`to aid solubilization. The solution was filter-sterilized and
`
`100 m1 of test solution dispensed into 250 ml flasks. Media
`without any parabcns were employed as controls. Before
`inoculation,
`the test solutions were maintained at
`their
`respective test temperatures in a shaking waterbath for at
`least 18 h.
`
`Test procedure
`
`In the experiments involving kill at various constant tem-
`peratures the test solutions were maintained at each tem-
`perature in separate shaking waterbaths (Grant SS40—D).
`In experiments involving a gradual increase in temperature
`over time the test solution was maintained in a Grant W14
`
`waterbath connected to a Grant temperature programmer
`P21. Since the Grant W14 waterbath is not a shaking
`waterbath, small sterile, magnetic teflon-coated fleas were
`included in the test solutions and were stirred on an lnspin
`2 (Baird and Tatlock). The temperature programmer was
`set to increase in temperature at the rate of 1°C/h, com-
`mencing at 34%: and rising to 42°C. This was closely mon-
`itored by a built-in thermometer and the inclusion of a
`thermometer in a control flask which was present in the
`waterbath.
`
`Measurement 01 mleroblal numbers
`
`After
`
`inoculation of the test solutions viable counts of
`
`microbial numbers were made at regular intervals. At each
`time interval a 1 m1 sample was removed from the test solu—
`tion 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.
`
`RESULTS AND DISCUSSION
`
`When exponential phase cells were placed in their growth
`medium the growth could be satisfactorily described by eqn
`
`.9
`
`..
`
`,.
`
`Lncfu/ml
`
`5
`
`16
`
`15
`
`‘Ti
`
`Time (min)
`
`Fig. 2 Comparison of the rate of growth of exponential phase
`Escherirhia mli cells in E], chemically defined media and 0,
`water. (Error bars are S.E. for 3 days' results.)
`
`(1) and a positive rate constant was obtained (Fig. 2). As
`expected, when the growth medium was substituted with
`water, no growth took place (Fig. 2). With 012% w/v
`methyl paraben and 0-012% w/v propyl paraben added to
`the growth medium bacterial kill was observed.
`The kill curve could be satisfactorily modelled by first
`order kinetics (Fig. 3) as shown by the linear semi-
`logarithmic plot of the number of surviving organisms
`against
`time. Figure 3 also shows that
`the rate of kill
`increased as the temperature increased. Table 1 lists the kill
`rate constants corresponding to the four different
`tem-
`peratures studied.
`
`Table 1 The efiect of temperature on the kill rate constants for
`inocula prepared from exponential and stationary phase
`Escherichia (all cells in chemically defined media in the presence
`of 012% w/v methyl paraben and 0-012% w/v propyl paraben
`
`Rate constant/h
`(mean ist.)
`
`Temperature(°C)
`
`Exponential
`phase cells
`
`Stationary
`phase cells
`
`—0-072 1 0-016
`—0-243 -_1- 0-026
`34
`--0-141 1 0-027
`—0-510 -1_- 0-008
`37
`—0-243 i 0-026
`— 1-546 i 0173
`40
`
`42 —3-442 -1_- 0-097
`
`
`
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`BACTERICIDAL EFFECT OF PARABENS 255
`
`
`'5
`
`I?
`
`[O
`
`Lncfu/ml
`
`0
`
`'00
`
`200
`
`300
`
`Time (min)
`Fig. 3 Rate ofkill ofan inoculum ofexponential phase cells of
`Escherichia mli in chemically defined media in the presence of
`0-12% methyl +0-012% propylparabens at D,34°C; .,37°C;
`I. 40°C and O, 42°C. (Error bars are st. for 3 days’ results.)
`
`The plot of the logarithm of the first order rate constant
`against the reciprocal of the temperature was linear (Fig. 4)
`thus showing that the Arrhenius equation provided a good
`model
`for
`the temperature effect on the kill
`rate and
`obviated the need to use more highly parameterized equa-
`tions or equations which are not transformed into a linear
`form. Simple linear regression provided an estimate for the
`activation energy of 274 kJ/mol.
`To investigate whether the activation energy was affected
`by the state in which the cells were in,
`the experiments
`were repeated using stationary phase cells instead of expo-
`nential phase cells. The data showed that the kill rate was
`first order (Fig. 5) and the Arrhenius plot was again linear
`(Fig. 6). The results were surprising in terms of how mark-
`edly different the kill rate constants for the stationary phase
`cells were from those relating to exponential phase cells
`(Table 1). The kill rate constants were in terms of hours for
`stationary phase cells and minutes for exponential phase
`cells. The activation energy (mean ist.) for the kill of
`stationary phase cells was low relative to exponential phase
`cells. 163 (34) kJ/mol vs 274 (17) kJ/mol- The practical
`implications of these observations are that stationary phase
`cells are much more resistant
`to the preservative com-
`bination at any given temperature than are exponential
`phase cells; a feature which must be borne in mind during
`pasteurization or sterilization processes.
`
`I?
`
`Lncfu/ml
`
`§
`
`\
`
` 7
`
`J:
`
`
`
`
`
`Lnrateconstantl/mln) l
`
`‘_l_
`
`
`
`—6 _l__l_i_1_t_l._._l_;—L_—l
`0-0032
`0-0032
`0-0032
`0-0032
`0-0032
`0-0033
`I/HKl
`Fla. 4 Arrhenius plot for the rate 0f kill of an inoculum of
`exponential phase Escherichia calf cells in chemically defined
`media in the presence of 012% methyl + 0-0l2% propyl
`parabens. (Error bars are 5.1;, n = 3)
`
`'
`|O
`
`0
`
`5
`
`20
`
`25
`
`30
`
`'5
`Time(hl
`Fig. 5 Rate of kill of an inoculum of stationary phase Escherichia
`coli cells in chemically defined media in the presence of 0-12%
`methyl +0-012% propyl parabens at El, 34°C; ., 37°C and I,
`40°C. (Error bars are S.E. for 3 days‘ results.)
`
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`

`—|-O
`
`—l-5
`
`--2-0
`
`—2-5
`
`—3-0
`
`!5
`
`Lnctu/rnl G
`
`|
`
`40
`
`l 34
`
`04an
`
`
`
`TemperaturePC)
`
`E E
`
`a EOU 9
`
`39
`
`C
`._l
`
`#5-5
`0.0032 @0032
`
`00032
`
`_I__l
`0-0033
`
`0-0032
`I/TlK)
`
`Fig. 6 Arrhenius plot for the rate of kill of an inoculum of
`stationary phase cells of Escherichia ME in chemically defined
`media in the presence of 0-l2% methyl +0.012% propyl
`parabens. (Error bars are S.E., n = 3.)
`
`O
`
`l 00
`
`300
`200
`Time (min)
`
`400
`
`500
`
`Fig. 7 Rate of kill of an inoculum of exponential phase
`Escherichia coli cells (+) in chemically defined media in the
`presence of 012% methyl + 0-012“/n propyl parabens during a
`gradual temperature increase from 34 to 42“C (0). (Error bars
`are 5.15., n = 3.)
`
`Comparison of our activation energies with those in the
`literature indicates that the high values observed,
`in our
`study with the parabens and E. coli, are of the same order
`of magnitude as those reported for other preservatives such
`as phenol, benzalkonium chloride and benzyl alcohol with a
`range of organisms
`(Table 2). Karabit
`at al.
`(1989),
`however, reported much more rapid kill than was observed
`in our studies. This can be explained by the fact that these
`authors used different preservative agents and experiments
`were carried out in standard phosphate buffers which are
`
`Table 2 Reported activation energies (Ea) for the effect of a
`series of antimicrobial agents
`____________—————
`Ea (kJ/mol)
`
`134
`65
`45
`Aspergill‘us m'gcr
`77
`150
`59
`Candida albicans
`86
`78
`93
`Escherichia coli
`76
`84
`72
`Pseudamonas aeruginasa
`92
`81
`91
`Staphylococcus aurcus
`________________———————
`
`a, 0-5% phenol at pH 6-1 in buffer (Karabit er al. 1985).
`b, 1% benzyl alcohol in pH 7-1 buffer (Karabit 2! all. 1986).
`c, 0001 % benzalkonium chloride in pH 6-1 butler except for Asp.
`niger for which 0-014“o benzalkonium chloride was used (Karabit
`et all. 1938).
`
`not expected to be suitable for growth. To investigate this
`aspect further the kill rate of exponential phase E. caIi was
`measured in sterile water where there was still no growth.
`Much more rapid kill was observed in this medium than in
`the growth medium. Indeed bacterial kill was so rapid that
`the temperature had to be lowered from the range 3H2°C
`to the range 26-5—340C to obtain practically measurable kill
`rates. At
`these lower temperatures the rates were l-OS/h
`(265°C), 2-15/h (290C), 3-86/h (31“C) and 5-25/h (34°C).
`The activation energy was 164 i 26 kJ/mol. Orth (1979)
`found, when calculating D—values for Staphylococcus auteur
`in a lotion, that the addition of brain heart infusion broth
`increased the D—value. It was suggested that
`the broth
`might have protected the cells by inactivating some of the
`preservative or that
`the broth supplied the bacteria with
`nutrients which allowed the organism to be less susceptible
`to the strain imposed by the preservative system.
`The results discussed so far show that, within the tem-
`perature range studied,
`the Arrhenius equation satisfact-
`orily modelled
`the
`kill
`rate
`constant—temperature
`relationship and interpolation within the range is justifiable.
`Under these conditions, non-isothermal kinetic modelling is
`often worthwhile. To investigate whether this approach,
`which has been used successfully to model chemical decom—
`position,
`is applicable to microbial kill kinetics we under—
`took a series of experiments in which the temperature of
`the preservative test medium was continuously altered.
`Figure 7 summarizes our findings.
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`BACTERICIDAL EFFECT OF PAHABENS 257
`__——_.—_.—._——-—n—-—_-—
`
`The temperature increase was linear with time but, as
`can be seen,
`the viable count showed increasingly large
`variability. Indeed that variability was too high to enable
`adequately precise rate constants to be calculated.
`It would therefore appear
`that although the non—
`isothermal method looked promising,
`the difficulty intro—
`duced by wide biological variation and low precision in
`bacterial enumeration,
`relative to chemical analysis, has
`contributed to making this method of little value in this
`instance.
`
`REFERENCES
`
`IRWIN, W.J., LI WAN Po, A. 51
`IIEMPENSTALL, ].M.,
`ANDREWS, A.H.
`(1983) Non-isothermal kinetics using a
`microcomputer: a derivative approach to the prediction of the
`stability of penicillin formulations. Journal of Pharmaceutical
`Sciences 72, 663—673.
`KARABIT, M.S.,
`JUNESKANS, OT. 8: L'L‘NDGREN, P.
`(I985) Studies on the evaluation of preservative efficacy I. The
`determination of antimicrobial characteristics of phenol. Arm
`Pharmaceutira Suerira 22, 2817290.
`Kama”, M.S.,
`JUNESKANS, 0.T. & LUNDGREN, P.
`(1986) Studies on the evaluation of preservative efficacy [1. The
`determination of antimicrobial characteristics of benzylalcohol.
`Journal ofClinical and Hospital Pharmacy ll, 281—289.
`Kano”, M.S.,
`JUNESKANS, O.T.
`8t LUNDGREN, P.
`(1988) Studies on the evaluation of preservative efficacy III.
`
`The determination of antimicrobial characteristics of benz-
`alkonium chloride. International Journal of Pharmacy 46,
`I41—
`147.
`
`JUNESKANS, 0.T. & LUNDGREN, P.
`KARABIT, M.S.,
`(1989) Studies on the evaluation of preservative efficacy IV.
`The determination of antimicrobial characteristics of some
`
`pharmaceutical compounds in aqueous solutions. International
`Journal ofPharmaceutics 54, 51v56.
`L1 WAN Po, A., ELIAS, A.N. & IRWIN, WJ. (1983)Non—
`isothermal and non-isopH kinetics in formulation studies. Aria
`Pharmaceutical Suecira 20, 2777286.
`LYNN, B. & HUGO, W.B. (1983) Chemical disinfectants, anti-
`septics and preservatives. In Pharmaceutical Microbiology, 3rd
`edn. Hugo, WB. 81 Russell, A.D. Oxford: Blackwell Scientific
`Publications.
`
`ORTH, D .S. (1979) Linear regression method for rapid detenni—
`nation of cosmetic preservative efficacy. Journal of the Society
`of Cosmetic Chemists 30, 3124432.
`PFLL‘G, 1.}. (1972) Heat sterilization. In Industrial Sterilisation:
`International Symposium, Amsterdam, 1972, Ch. 14. ed. Phillips,
`GB. 8: Miller, W.S. North Carolina: Duke University Press.
`Rarxowsxr, D.A., Lower, R.K., MCMEEKIN, T.A.,
`STOKES, A.N. 81 CHANDLER, R.E. (1983) Models for bac-
`terial culture growth rate throughout the entire biokinetic tem-
`perature range. Journal of Bacteriology 154, 122271226.
`SCHOOLFIELD, R.M., SHARPE, P.J.H.
`Sc MAGNUSON,
`C.E.
`(1931) Non-linear regression of biological temperature-
`dependcnt rate models based on absolute reaction—rate theory.
`Journal of Theoretical Biology 88, 719—731.
`
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