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Neutralisation of the antibacterial action of quaternary ammonium compounds with cyclodextrins
FEMS MicrobiologyLetters 90 (1992) 197-2(~1 x~ 1992 Federation (if European Microbiok~gicalSocieties (1378-1097/:;2/%',~5.'~'~ Published by Elsevier
FEMSLE 1147511
Neutralisation of the antibacterial action of quaternary ammonium compounds with cyclodextrins W.J. S i m p s o n Brewing Research [.'oundation. Nutfi','kL Surrc)'. U.K.
Rt:cei~cd 7 October lt)ql Accepted 22 October Iqt91
Key words: Cyclodextrin: Neutralisation: Quaternary ammonium compcmnd
1. S U M M A R Y Cyclodextrins can be used to inactivate the antimicrobial activity of quaternary ammonium biocides. Their use offers several advantages over other methods which employ non-ionic detergents or phospholipids.
2. I N T R O D U C T I O N When organisms from media containing antibiotics or disinfectants are isolated, it is common practice to arrest antimicrobial activity by use of a neutralising agent. For example. Tween 80 (polyoxyethyleoe sorbitan mono-oleate) is used to inactivate the antimierobial activity of benzoic
acid and sorbic acid and mixtures of Tween 80 and lecithin, or Lubrol W and lecithin are used to inactivate quaternary ammonium compounds (QACs) [1,2]. in such cases, molecules of the antimicrobial agent become trapped in micelies of the neutralising agent, thereby preventing them from interacting with microbial cells. Neutralisers of this type, however, can bc lethal to some bacteria [3]. Although high concentrations of Tweens and other detergents antagonise the action of QACs, low concentrations can potentiate their action [4]. This paper reports that cyclodextrins (CDs) provide an alternative to detergents for neutralisation of antibacterial QACs.
3. M A T E R I A L S A N D M E T H O D S
3.1. C h e m i c a l s Correspondence to: W.J. Simpson, Brewing Research Founda-
tion, Nutfield. Surrey RHI 4HY. U.K.
Quaterpary ammonium coumpounds (QACs) and cyelodextrins (CDs) were obtained from Sigma (U.K.).
Iq8
3.2. Assays of antibacterial acti('ity Bacillus subtilis ATCC 6633 was grown in MYGP medium (malt extract 3 g; yeast extract 3 g; glucose 10 g; peptone 5 g; de-ionised water to 1000 ml) at 25°C for 16 h. Assays of minimum inhibitor3, concentration (MIC) were performed using inocula of 1 x 105 organisms/ml in MYGP or MYGP supplemented with a-CD or/3-CD (10 mM; sterile-filtered prior to use). A range of doubling dilutions of benzethonium chloride was tested in each medium. Growth was assessed after a 48-h incubation at 25°C. In addition, paper strip neutralisation assays [5] were carried out using B. subtilis and MYGP medium solidified with agar (15 g/I). One paper strip was impregnated with benzethonium chloride solution (2 mM in water), the other with either a-CD or fl-CD solution (10 mM in water). 3.3. Critical micelle concentrations (CMCs) CMCs were determined from plots of surface tension vs. log concentration. The surface tension of benzethonium chloride solutions was measured at 21°C in 0.1 M sodium 3,3'-dimethylglutarate buffer (pH 7.0), in the presence or absence of 1 mM /3-CD, using DuNouy tensitometer (Cambridge Instruments, U.K.).
4. RESULTS
4.1. Minimum inhibitory concentrations (MiCs) In the absence of CDs the MIC of benzethonium chloride for B. subtilis ATCC 6633 was 0.78 p.M. In the presence of 10 mM o~-CD o r / 3 - C D the MIC was 1.56 /.tM and 1.0 mM respectively. The difference between the control and a-CD results was not significant. 4.2. Paper strip diffusion assays ot-CD did not affect the size of the inhibition zone produced by benzethonium chloride; /3-CD increased the zone size by approximately 2-fold. 4.2. Critical micelle concentrations (CMCs) In the absence of CDs the CMC of benzethonium chloride in 0.1 M sodium, 3,3'-dimethyl-
glutarate buffer (pH 7.0) was 0.1 mM. In the presence of/3-CD (1 raM) the CMC was 1.7 mM.
5. DISCUSSION Cyclodextrins are obtained as a result of the action of cyclodextrin transferases on starch, aCD, B-CD and 3,-CD are composed of six, seven and eight glucose residues respectively, the molecules being arranged such that the exterior of the CD is hydrophilic but the interior is hydrophobic. CDs form inclusion complexes with a number of organic compounds (for review see ref. 6). Such 'guest' compounds become partially or completely trapped within the hydrophobic cavity of the CD molecule. The ability to form a complex depends on the size and polarity of the guest compound and the size of the CD cavity. The fact that CDs fotm inclusion complexes with QACs such as benzethonium chloride is indicated by the increased CMC of benzethonium chloride in the presence of /3-CD; and by the increased solubility of benzethonium chloride in the presence of/3-CD (results not shown), a-CD does not form a complex with benzethonium chloride. As a consequence of complex formation /3-CD inactivates the antibacterial activity of benzethonium chloride, the presence of 10 mM/3-CD increasing the MIC of this compound almost 1000-fold. In agar diffusion assays, the phenomenon of inclusion complex formation can be used to increase assay sensitivity. In general, miceiles of Q A C would be expected to diffuse more slowly through agar media than would individual molecules. Presumably, the suppression of micellisation induced by the presence of /5-CD enhances the diffusion rate of the Q A C in the agar medium thus enlarging the inhibition zone. Complex formation can be utilised to inactivate the antimicrobial activity of a wide range of QACs. For example, a-CD will inactivate benzalkonium chloride and cetyl trimethyl ammonium bromide; /3-CD will inactivate benzethonium chloride, methylbenzethonium chloride, benzalkonium chloride and cetyl trimethyl ammonium
199 bromide; 7 - C D will inactivate b e n z e t h o n i u m chloride a n d ~ r n e t h y l b e n z e t h o n i u m chloride. T h e use of C D s for neutralisation of Q A C s offers several a d v a n t a g e s over t h e d e t e r g e n t s referred to earlier. C D s generally have no effect on microbial growth or m e t a b o l i s m , arc not surfaceactive, are not m e t a b o l i s e d by m i c r o o r g a n i s m s , are very soluble in aqueou:~ m e d i a a n d t h e r e f o r e provide a high d e g r e e o f neutralisation capacity, arc stable in solution (solutions with a p H value b e t w e e n 2 a n d 12 can be stored at r o o m t e m p e r a ture for several years without significant deterioration) a n d are inexpensive a n d available from a wide r a n g e of suppliers. N e u t r a l i s a t i o n can be m a d e selective by c h o o s i n g an :~ppropriate C D for inactivation of o n e or m o r e Q A C s or, alternatively, two or m o r e C D s c a n be used to n e u t r a l i s t several Q A C s . C D s s h o u l d prove useful in a wide ranbc of applications w h e r e n e u t r a l i s a t i o n of q u a t e r n a r y a m m o n i u m biocides is necessary. It s h o u l d also be possible to neutralise o t h e r antimicrobial a g e n t s in this way.
ACKNOWLEDGEMENTS 1 t h a n k Dr A . R . W . Smith for helpful advice a n d the Director of the Brewing R e s e a r c h F o u n dation for permission to publish.
REFERENCES
[I] Anon. (1960) Methods fi~r the laboratory evaluation of the disinfectant activity of quaternary ammonium compounds, British Standard 32X6. London: IIMSO. [21 Russell. A.D.. Ahonkai, I. and Rogers. D.T. (1979) J. Appl. Bacteriol. 47. 207-245. [3] Laycock, It.tl. and Mulley. B.A. 11970)J, Pharm. Pnarmacol. 22. 157-162. [4] AIIwood. M.C. (1973~ Microbios 7. 203-214. [5] Bcale. A.S. and Sulherhmd. R. (1983) In: Antibiotics: Assessment of Antlmicr,,;bial Activity and Resistance. (Russell, A.D. and Quesncl. LB.. Eds.). pp. 229-315. Society Ihr Applied Bacterial Technology Serial No. 18. Academic Press. London. [6] Szejtli. J. (198N) Cyclodextrin Technology. 4.5i~pp. Klawcr Academic Pui31ishcrs.Dordrecht. The Netherlands.
FEMSLE 1147511
Neutralisation of the antibacterial action of quaternary ammonium compounds with cyclodextrins W.J. S i m p s o n Brewing Research [.'oundation. Nutfi','kL Surrc)'. U.K.
Rt:cei~cd 7 October lt)ql Accepted 22 October Iqt91
Key words: Cyclodextrin: Neutralisation: Quaternary ammonium compcmnd
1. S U M M A R Y Cyclodextrins can be used to inactivate the antimicrobial activity of quaternary ammonium biocides. Their use offers several advantages over other methods which employ non-ionic detergents or phospholipids.
2. I N T R O D U C T I O N When organisms from media containing antibiotics or disinfectants are isolated, it is common practice to arrest antimicrobial activity by use of a neutralising agent. For example. Tween 80 (polyoxyethyleoe sorbitan mono-oleate) is used to inactivate the antimierobial activity of benzoic
acid and sorbic acid and mixtures of Tween 80 and lecithin, or Lubrol W and lecithin are used to inactivate quaternary ammonium compounds (QACs) [1,2]. in such cases, molecules of the antimicrobial agent become trapped in micelies of the neutralising agent, thereby preventing them from interacting with microbial cells. Neutralisers of this type, however, can bc lethal to some bacteria [3]. Although high concentrations of Tweens and other detergents antagonise the action of QACs, low concentrations can potentiate their action [4]. This paper reports that cyclodextrins (CDs) provide an alternative to detergents for neutralisation of antibacterial QACs.
3. M A T E R I A L S A N D M E T H O D S
3.1. C h e m i c a l s Correspondence to: W.J. Simpson, Brewing Research Founda-
tion, Nutfield. Surrey RHI 4HY. U.K.
Quaterpary ammonium coumpounds (QACs) and cyelodextrins (CDs) were obtained from Sigma (U.K.).
Iq8
3.2. Assays of antibacterial acti('ity Bacillus subtilis ATCC 6633 was grown in MYGP medium (malt extract 3 g; yeast extract 3 g; glucose 10 g; peptone 5 g; de-ionised water to 1000 ml) at 25°C for 16 h. Assays of minimum inhibitor3, concentration (MIC) were performed using inocula of 1 x 105 organisms/ml in MYGP or MYGP supplemented with a-CD or/3-CD (10 mM; sterile-filtered prior to use). A range of doubling dilutions of benzethonium chloride was tested in each medium. Growth was assessed after a 48-h incubation at 25°C. In addition, paper strip neutralisation assays [5] were carried out using B. subtilis and MYGP medium solidified with agar (15 g/I). One paper strip was impregnated with benzethonium chloride solution (2 mM in water), the other with either a-CD or fl-CD solution (10 mM in water). 3.3. Critical micelle concentrations (CMCs) CMCs were determined from plots of surface tension vs. log concentration. The surface tension of benzethonium chloride solutions was measured at 21°C in 0.1 M sodium 3,3'-dimethylglutarate buffer (pH 7.0), in the presence or absence of 1 mM /3-CD, using DuNouy tensitometer (Cambridge Instruments, U.K.).
4. RESULTS
4.1. Minimum inhibitory concentrations (MiCs) In the absence of CDs the MIC of benzethonium chloride for B. subtilis ATCC 6633 was 0.78 p.M. In the presence of 10 mM o~-CD o r / 3 - C D the MIC was 1.56 /.tM and 1.0 mM respectively. The difference between the control and a-CD results was not significant. 4.2. Paper strip diffusion assays ot-CD did not affect the size of the inhibition zone produced by benzethonium chloride; /3-CD increased the zone size by approximately 2-fold. 4.2. Critical micelle concentrations (CMCs) In the absence of CDs the CMC of benzethonium chloride in 0.1 M sodium, 3,3'-dimethyl-
glutarate buffer (pH 7.0) was 0.1 mM. In the presence of/3-CD (1 raM) the CMC was 1.7 mM.
5. DISCUSSION Cyclodextrins are obtained as a result of the action of cyclodextrin transferases on starch, aCD, B-CD and 3,-CD are composed of six, seven and eight glucose residues respectively, the molecules being arranged such that the exterior of the CD is hydrophilic but the interior is hydrophobic. CDs form inclusion complexes with a number of organic compounds (for review see ref. 6). Such 'guest' compounds become partially or completely trapped within the hydrophobic cavity of the CD molecule. The ability to form a complex depends on the size and polarity of the guest compound and the size of the CD cavity. The fact that CDs fotm inclusion complexes with QACs such as benzethonium chloride is indicated by the increased CMC of benzethonium chloride in the presence of /3-CD; and by the increased solubility of benzethonium chloride in the presence of/3-CD (results not shown), a-CD does not form a complex with benzethonium chloride. As a consequence of complex formation /3-CD inactivates the antibacterial activity of benzethonium chloride, the presence of 10 mM/3-CD increasing the MIC of this compound almost 1000-fold. In agar diffusion assays, the phenomenon of inclusion complex formation can be used to increase assay sensitivity. In general, miceiles of Q A C would be expected to diffuse more slowly through agar media than would individual molecules. Presumably, the suppression of micellisation induced by the presence of /5-CD enhances the diffusion rate of the Q A C in the agar medium thus enlarging the inhibition zone. Complex formation can be utilised to inactivate the antimicrobial activity of a wide range of QACs. For example, a-CD will inactivate benzalkonium chloride and cetyl trimethyl ammonium bromide; /3-CD will inactivate benzethonium chloride, methylbenzethonium chloride, benzalkonium chloride and cetyl trimethyl ammonium
199 bromide; 7 - C D will inactivate b e n z e t h o n i u m chloride a n d ~ r n e t h y l b e n z e t h o n i u m chloride. T h e use of C D s for neutralisation of Q A C s offers several a d v a n t a g e s over t h e d e t e r g e n t s referred to earlier. C D s generally have no effect on microbial growth or m e t a b o l i s m , arc not surfaceactive, are not m e t a b o l i s e d by m i c r o o r g a n i s m s , are very soluble in aqueou:~ m e d i a a n d t h e r e f o r e provide a high d e g r e e o f neutralisation capacity, arc stable in solution (solutions with a p H value b e t w e e n 2 a n d 12 can be stored at r o o m t e m p e r a ture for several years without significant deterioration) a n d are inexpensive a n d available from a wide r a n g e of suppliers. N e u t r a l i s a t i o n can be m a d e selective by c h o o s i n g an :~ppropriate C D for inactivation of o n e or m o r e Q A C s or, alternatively, two or m o r e C D s c a n be used to n e u t r a l i s t several Q A C s . C D s s h o u l d prove useful in a wide ranbc of applications w h e r e n e u t r a l i s a t i o n of q u a t e r n a r y a m m o n i u m biocides is necessary. It s h o u l d also be possible to neutralise o t h e r antimicrobial a g e n t s in this way.
ACKNOWLEDGEMENTS 1 t h a n k Dr A . R . W . Smith for helpful advice a n d the Director of the Brewing R e s e a r c h F o u n dation for permission to publish.
REFERENCES
[I] Anon. (1960) Methods fi~r the laboratory evaluation of the disinfectant activity of quaternary ammonium compounds, British Standard 32X6. London: IIMSO. [21 Russell. A.D.. Ahonkai, I. and Rogers. D.T. (1979) J. Appl. Bacteriol. 47. 207-245. [3] Laycock, It.tl. and Mulley. B.A. 11970)J, Pharm. Pnarmacol. 22. 157-162. [4] AIIwood. M.C. (1973~ Microbios 7. 203-214. [5] Bcale. A.S. and Sulherhmd. R. (1983) In: Antibiotics: Assessment of Antlmicr,,;bial Activity and Resistance. (Russell, A.D. and Quesncl. LB.. Eds.). pp. 229-315. Society Ihr Applied Bacterial Technology Serial No. 18. Academic Press. London. [6] Szejtli. J. (198N) Cyclodextrin Technology. 4.5i~pp. Klawcr Academic Pui31ishcrs.Dordrecht. The Netherlands.