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The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces
FEMS Microbiology Ecology Published by Elsevier
45 (1987) 297-304
297
FEC 00134
The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces Martyn
Humphries,
Jozef
Nemcek,
John
B. Cantwell
and John
Corporate Collard Science Group, ICI PLC, Runcorn, Cheshve,
J. Gerrard
U.K.
Received 24 February 1987 Revision received 2 June 1987 Accepted 9 June 1987
Key words:
Polyethylene
glycol (PEG) graft copolymers;
1. SUMMARY Twelve graft copolymers have been evaluated for their ability to prevent the adhesion of bacteria to substrata. The copolymers had polyethylene glycol (PEG) side-chains (‘teeth’) and a backbone that was either uncharged, acidic, basic or amphoteric. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite substrata, and 2-h petri-dish adhesion experiments performed with bacteria isolated from marine (Pseudomonas sp. NCMB 2021) paper mill (Smarcescens NCIB 12211) and oral (Smutans NCTC 10449) environments. The copolymers containing the most charged groups in the backbone had the most significant effect on bacterial adhesion levels, with anti-adhesive effects up to 99% achieved. An amphoteric copolymer (Compound 12) on glass, and acidic copolymer (Compound 11) on stainless steel and hydroxyapatite gave the most impressive anti-adhesive effects. These copolymers had nonspecific bacterial anti-adhesive properties.
Correspondence to: M. Humphries, Corporate Colloid Science Group, ICI PLC. P.O. Box 11, The Heath, Runcorn, Cheshire WA7 4QE. U.K. 016%6496/87/$03.50
0 1987 Federation
of European
Microbiological
Anti-adhesive
compounds;
Hydrophilic
substrata
It is proposed that the graft copolymers adsorbed onto hydrophilic surfaces via their charged backbone in such a way that the PEG side-chains were pointing out into the aqueous phase, and it was this orientation that was responsible for the observed anti-adhesive effect.
2. INTRODUCTION Bacterial adhesion to solid surfaces [1,2] is a universal and usually unwanted phenomenon which can be considered to be the primary event, for example, in dental plaque formation [3], marine fouling [4,5] and biofilm formation in circulating water systems [6]. Previous papers [7,8] have shown that the adsorption of ethoxylated (and propoxylated) surfactants onto hydrophobic, but not hydrophilic substrata resulted in significant reductions in bacterial adhesion levels in laboratory experiments. This ‘anti-adhesive’ effect was believed to be due to the specific mode of adsorption of linear ethoxylated surfactants on hydrophobic (as opposed to hydrophilic) substrata, i.e. via their hydrophobe in such a way that the polyethylene glycol (PEG) chains were pointing out into the aqueous phase. It was proposed that the hydroSocieties
298
philic, highly hydrated PEG chains provided a steric barrier which prevented the adhesion of bacteria. We suggested that a certain density of PEG chains at the interface would be necessary to achieve the anti-adhesive effect. It was proposed that in theory it should be possible to render any surface anti-adhesive assuming that molecules could be adsorbed and oriented in such a way as to give a high density of PEG chains at the interface. The objective of the experiments described in this paper was to test this hypothesis. The main difficulty was seen to be achieving the adsorption of hydrophilic molecules onto a hydrophilic substratum in an aqueous environment. We believed that this was most likely to occur with polymeric molecules, which having a multiplicity of anchoring points should be more substantive to a surface than the corresponding monomeric compounds. Thus we have synthesised a number of graft copolymers which have a ‘backbone’ which can be uncharged, acidic, basic or amphoteric, with PEG chains as ‘teeth’. The design intention was that the backbone would interact with the substratum in such a way that the PEG chains would point out into the aqueous phase, resulting in an anti-adhesive surface. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite surfaces. Their effects on the adhesion of a marine bacterium (Pseudomonas sp NCMB 2021) paper mill bacterium (S.marcescens NCIB 12211) and an oral bacterium (S.mutans NCTC 10449) were investigated.
3. METHODS 3.1. Synthesis of graft copolymers The graft copolymers (‘combs’) were made by free radical copolymerisation of cu-methoxy-wmethacroyl polyethylene glycols (‘teeth’) with various combinations of methyl methacrylate (MMA)(CH, = C(CH,)CO,CH,), methacrylic acid (MAA) (CH, = C(CH,)CO,H) and N,N-dimethyl-2 aminoethyl methacrylate (DMAEM) (CH, = C(CH,)CO,CH,CH,N(CH,),) in the backbone. 3.1.1. Monomers. Methyl methacrylate (ICI Mond Division), methacrylic acid (BDH) and
IV, N-dimethylaminoethyl methacrylate (Aldrich) were distilled under reduced pressure and stored at 4°C before use. Methoxy polyethyleneglycol methacrylates (for Compounds l-4) were prepared using the general method described below: Freshly distilled methacroyl chloride (0.11 mol) was slowly added to a 30% w/v solution of 2,6-dimethyl pyridine (0.11 moles) [2,6-lutidine (BDH), which was distilled under reduced pressure before use] in toluene, in a 3-necked round bottom flask. The flask was equipped with a dropping funnel, magnetic stirrer, nitrogen bleed and condenser. Some fuming occurred during addition, and a white precipitate formed. The mixture was cooled in an ice bath and methoxy-ended polyethyleneglycol [dried by bubbling nitrogen through the heated liquid (115 “C) under reduced pressure] (0.10 mol, Aldrich) was added dropwise, with stirring, under a nitrogen blanket. A copious white precipitate formed as the reaction proceeded. The addition took 3 h, and on completion the reaction mixture was stirred on ice for an additional 2 h, before the reaction flask was allowed to reach room temperature. The precipitated 2,6-lutidine hydrochloride was filtered off and washed with small amounts of toluene. The combined filtrate was dried down on a rotary evaporator at 45°C under reduced pressure. The product (89-97s yield) was analysed by infra-red, nuclear magnetic resonance and by vinyl bond titration. The methoxy polyethyleneglycol methacrylates were stored at 4 o C before use. Methoxypolyethyleneglycol methacrylate 400 (= nominal M, - Polysciences) was used as obtained for Compounds 5-12. Nuclear Magnetic Resonance spectroscopy revealed the M, to be approximately 420, i.e. the polyethyleneglycol chain had a M, of approximately 350. 3.1.2. Polymerisations. A general method is described below: Free radical polymerisations were performed at 70” C in a 600-ml reaction flask equipped with nitrogen bleed, water cooled condenser, thermometer and stirrer. The reaction vessel was placed in a thermostatically controlled oil bath. Monomers in selected molar ratios (total 25 g) were added together with 300 ml of solvent
299
(toluene or industrial methylated spirits/water mixtures) and heated to 70 “C. An aliquot of the initiator (1.8 x 1O-3 mol) 2,2’-azobis (2-methylpropionitrile) (BDH) or 4,4’-azobis (4cyanovaleric acid) (Aldrich) in 20 ml solvent was added and the mixture polymerised with stirring at 200 rpm for 24 h. A second aliquot of initiator (7 X lop4 mol) in 20 ml solvent was then added and the polymerisation continued for a further 24 h. The copolymers were isolated by evaporation under high vacuum of volatile materials (residual starting materials) present in the reaction mixture. The theoretical average compositions of the copolymers were largely confirmed by chemical analysis, nuclear magnetic resonance and acid-base titrations. The average composition of the copolymers are given in Table 1. An example of the structure of a copolymer containing MMA, MAA and DMAEM is given below:
(CH,CH,O),CH, MMA
MA.4
DMAEM
PEG Methacrylate
The M,s of the copolymers were determined by gel permeation chromatography in dimethylformamide-lithium bromide system, using polyethylene oxide as standard. The A4,s were in the range 35 000-70 000 and polydispersities (M,/Mn) 1.5-3 (typical values for free radical polymerisations). Due to the lack of suitable standards the M, values should only be taken as a guideline. 3.1.3. Polymethactylic acid and poly DMAEM. An aqueous solution of polymethacrylic acid (PMMA) of M, 25000 was obtained from Allied Colloids (Versicol-K13). DMAEM Poly (PDMAEM) of M, approximately 30 000 was prepared in our laboratory. 3.2. Organisms and growth conditions A marine Pseudomonas sp. strain National Collection of Marine Bacteria 2021 (NCMB 2021) was stored on Marine Agar plates and grown up in 100 ml batches of Marine Broth (Difco) in a
G24 Environmental Incubator Shaker (New Brunswick) at 18” C, 200 rev/mm for 17 h. The bacteria were diluted 10 times in 2.4% w/v saline for adhesion experiments. The bacterial concentration was approximately 5 X 106. ml-’ (determined by counting in a haemacytometer). Serratia marcescens strain National Collection of Industrial Bacteria 12211 (NCIB 12211) was stored on Nutrient Agar plates and grown up in 100-ml batches of Nutrient Broth (Oxoid) in the incubator shaker at 30°C, 200 rev/min for 17 h. The bacteria were diluted 10 times in distilled water for adhesion experiments. The bacterial concentration was approximately 1 X lo6 f ml-’ (determined by counting in a haemacytometer). Streptococcus mutans strain National Collection of Type Cultures 10449 (NCTC 10449) was stored on Brain Heart Infusion Agar plates and grown up in loo-ml batches of Brain Heart Infusion Broth (Oxoid) in the incubator shaker at 37” C, 200 rev/mm for 17 h. The bacteria were centrifuged for 10 min at 4000 rev/mm, resuspended in modified Ringer’s salts solution (0.54 g. 1-l NaCl, 0.02 g.l-’ KCl, 0.03 g.ll’ CaCl,, 0.75 g.ll’ sodium mercaptoacetate), recentrifuged, resuspended and diluted 10 times in modified Ringer’s salts solution. The bacterial concentration was approximately 1 X lo6 . ml-’ (determined by counting in a haemacytometer). 3.3. Surfaces of attachment Glass microscope slides, stainless steel discs and hydroxyapatite discs were used as substrata for bacterial adhesion experiments. Glass microscope slides were cut into thirds, immersed in methanol for 10 min, rinsed and stored in distilled water before use. Stainless steel discs were made from a 25-mm rod by parting off 2 mm thick sections on a lathe. The lathe marks were removed using silicone carbide paper on a lapping machine, and the discs given a mirror polish using a micro-cloth and 6-pm diamond resuspended in water. The discs were made hydrophilic by immersing in boiling 50% phosphoric acid for 2 min. The discs were thoroughly rinsed and stored in distilled water before use. After an experiment, the discs were repolished and reheated in acid for re-use. Hy-
300
Table
1
Average
composition
Copolymer number
Backbone nature
1 2 3 4 5 6 7 8 9 10 11 12
Acid Acid Base Base Acid Base Amphoteric Acid Uncharged b Base b Acid b Amphoteric
of the graft copolymers Side-chain
Molar ratios a
(‘+f,)
I
PEG 550 PEG2000 PEG 550 PEG2000 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350
4:l 13:l 4:l 1O:l 4:l 4:l 2.7:1 2:l 2.7 : 1 3 :1 3:l 3:l
II (MMA : MAA DMAEM)
:
1O:l:O 1O:l:O 18:O:l 18:O:l 1O:l:O 18:O:l 36:1.8:1 1:l:O All MMA All DMAEM All MAA 0:1.8:1
a Molar ratio I = No. of monomer units in backbone: No. of side-chains (not including the methacrylate component of the side-chains). Molar ratio II = ratio of monomers in the backbone (not including the methacrylate component of the side-chains). b No MMA in the backbone.
droxyapatite discs were made by compressing hydroxyapatite powder (calcium phosphate tribasic [Ca,,(OH),(PO,),] (Aldrich) in a 3-cm evacuable X-ray die (Specac, Orpington, U.K.). The discs were heated for 4 h in a furnace at 1100 o C. Hydroxyapatite discs were re-used after heating for 2 h at 900°C. The discs were equilibrated in distilled water overnight prior to bacterial adhesion experiments. 3.4. Bacterial adhesion experiments Glass microscope slides, stainless steel discs and hydroxyapatite discs were immersed in 1% w/v aqueous solutions of the copolymers (also PMAA and PDMAEM) at ambient temperature for 5 min. The slides and discs were given a standard wash by immersing and shaking 5 times in a container of flowing water, and placed in polystyrene petri-dishes. 30 ml of bacterial suspenof Pseudomonas sp. (NCMB 2021) sion Xmarcescens (NCIB 12211) or Xmutans (NCTC 10449) was added to each petri-dish. After 2 h at ambient temperature, the slides and discs were taken out of the bacterial suspension and loosely
attached bacteria removed by immersing and shaking 5 times in a container of flowing water. Adhered bacteria were stained using Loeffler’s Methylene Blue (0.3 g methylene blue, 30 ml 90% ethanol, 100 ml water). Computerised image analysis (Kontron Image Process System) was used to measure bacterial adhesion levels [9]. Percentage adhesion of copolymer-treated substrate relative to untreated controls was calculated = %antiadhesion (0% anti-adhesion = control, 100% antiadhesion = no adhered bacteria). The experiments were performed twice, and mean values calculated.
4. RESULTS Table 2 gives the percentage surface area of substrata (glass, stainless steel and hydroxyapatite) covered by adhered bacteria (= % adhesion) after 2 h petri-dish adhesion experiments with Pseudomonas sp. (NCMB 2021) Xmarcestens (NCIB 12211) and Smutans (NCTC 10449). Tables 3, 4 and 5 show the effect of adsorbing the graft copolymers onto glass, stainless steel and hydroxyapatite substrata prior to 2 h petri-dish adhesion experiments. The results are expressed as % reduction in bacterial adhesion levels compared to untreated controls (= % anti-adhesion). Mean values of duplicate experiments are given.
5. DISCUSSION
The graft copolymers (Table 1) can be conveniently divided into those which contain MMA in
Table 2 Percentage surface area covered by adhered bacteria after 2 h petri-dish adhesion experiments with Pseudomonas sp. (NCMB 2021), Smarcescens (NCIB 12211) and S.mutans (NCTC 10449) %Adhesion
Glass Stainless steel Hydroxyapatite
Pseudomonas
S.mnrcescens
6.8
9.1
S.mutans 18.1
4.2 3.9
7.7 3.6
28.0 27.9
301 Table 3
Table 5
The effect of adsorbing graft copolymers onto glass, stainless steel and hydroxyapatite surfaces on the adhesion of Pseudomonas sp. (NCMB 2021) during 2 h petri-dish adhesion experiments
The effect of adsorbing graft copolymers onto glass, stainless steel and hydroxyapatite surfaces on the adhesion of Xmutons (NCTC 10449) during 2 h petri-dish adhesion experiments.
Mean value of duplicate experiments and the spread of results is given. Graft copolymer
Glass
PMAA PDMAEM
Hydroxyapatite
Stainless steel
47* 4 +63+10
30+ 32k 35k 1+ 17* 33i 28+ 32* 185 6* 95+ 75*
l+ 8 24k 0 35* 7 8+14 14* 4 37+ 6 21& 4 34+ 2 43* 1 81k 3 96+ 2 81+ 3
+9* 6 14*10 71rfr 4 20+ 1 24+ 8 43+10 52* 7 54* 4 52k 5 97+ 1 85+ 2 98k 1
1 2 3 4 5 6 7 8 9 10 11 12
+
% Anti-adhesion
28* +31* -
1 3 4 9 9 4 2 2 7 5 1 4
70& 2 +95*17 -
7 4
= Increase in bacterial adhesion levels.
Table 4 The effect of adsorbing graft copolymers onto glass, stainless steel and hydroxyapatite surfaces on the adhesion of S.marcescens (NCIB 12211) during 2 h petri-dish adhesion experiments Mean value of duplicate experiments and the spread of results is given. Graft copolymer
% Anti-adhesion Glass
Hydroxyapatite
12*
+10*
I
23k
2 3 4 5 6 7 8
30* 4 74* 0 37* 2 21* 2 49+10 37+ 6 24+ 8
29+10 17* 7 lo+ 2 15+ 1 26kll 3*10 9+ 6
9 10 11 12
43+ 99* 86k 99+
4 1 3 1
lo* 90+ 95* 55*
2 2 1 4
PMAA PDMAEM
17* 75*
4 2
22+ 68?
8 5
+
6
Stainless steel 5
= Increase in bacterial adhesion levels.
5
20+12 8k 5 18k 4 21+ 4 +4* 5 53+ 9 +14*16 52+ 23k 94+ 64k
2 6 2 5
77+ 1 +23+11
Mean value of duplicate experiments and the spread of results is given. Graft copolymer
% Anti-adhesion Stainless steel
Glass
1 2 3 4 5
58+ 15& 75+ 55+ 55+
1 6 4 1 3
11+ +I+13 28+ 11* 6&
6
345
2
+11*11
7 8 9
30* 7 38+10 52+ 4 11* 91+ 98k
10 11 12 PMAA PDMAEM
+
3 1 1
7*11 +38+ 5
17* 24* +15* 17* 94i_ 47*
Hydroxyapatite
6 2 8
165 +2+ +fi* 18k +105
6 I 7 3 9
8k
2
5 7 7
13* 32* 6k
1 8 9
4 4 5
21* 96+ 68k
2 1 4
3
17* 7 +46+12
71+ 2 +34+10
= Increase in bacterial adhesion levels.
the backbone (Compounds l-9) and those which do not (Compounds 10-12). Of the former group, Compounds l-7 contain mostly MMA with only a few acidic (MM) and/or basic (DMAEM) groups in the backbone. Compound 8 contains approximately equal numbers of MMA and acidic groups, while Compound 9 is composed of only MMA, i.e. no charged groups. In contrast, Compounds lo-12 have all acidic and/or basic groups in the backbone. The results in Tables 3, 4 and 5 show that it was the latter group that gave the most impressive anti-adhesive effects. The design intention of the graft copolymers was that the charged backbone would interact with the substratum in such a way that there was a high density of PEG chains pointing out into the aqueous phase, and that this orientation was responsible for the anti-adhesive effect, i.e. we have somewhat simplistically divided the copolymers into an effect end (PEG side-chains) and adsorbing end (backbone). However, the charged groups in the backbone of the graft copolymers could also be a factor in bacterial adhesion. For this reason, the effect of a standard acidic and basic polymer
302
was investigated. Polymethacrylic acid (PMAA) and poly-N, N-dimethylaminoethyl methacrylate (PDMAEM) were chosen. They could be considered to be Compounds 10 and 11, respectively, without the PEG chains. Results in Tables 3, 4 and 5 show that PMAA and PDMAEM do have some effect on bacterial adhesion levels. PMAA gave approximately 70% anti-adhesive effect with all 3 bacteria on hydroxyapatite, but little effect on glass and stainless steel. PDMAEM actually increased bacterial adhesion levels with Pseudomonas sp. (NCMB 2021) and S.mutans (NCTC 10449) on all 3 surfaces, and with Smarcescens (NCIB 12211) on hydroxyapatite. However, PDMAEM gave approximately 70% anti-adhesive effect with Xmarcescens (NCIB 12211) on glass and stainless steel. Thus for the PEG chains of the graft copolymers to be having a significant role in preventing bacterial adhesion, it was considered necessary to obtain anti-adhesive effects well in excess of the 70% level. Anti-adhesive levels of 90-95s or greater were somewhat arbitrarily considered to be significant. Tables 3, 4 and 5 show that of Compounds 1-9, only Compound 6 yields anti-adhesive effects at the 70% level, and this was only achieved on glass. These anti-adhesive effects were not considered to be significant. In contrast, Compounds 10, 11 and 12 gave anti-adhesive effects both within and above the 90-95% level, even as high as 99% anti-adhesion. The results in Tables 3, 4 and 5 demonstrate both bacterial and substratum specificity. The major observations are listed below. Compound 10 (basic). (1) No significant antiadhesive effect with all 3 bacteria on hydroxyapatite, or with S.mutans (NCTC 10449) on all 3 surfaces. (2) Yields greater than 95% anti-adhesion with Pseudomonas sp. (NCMB 2021) and S.murcescens (NCIB 12211) on glass. This was reduced to 80-90s anti-adhesion on stainless steel. Compound II (acidic). (1) Approximately 95% anti-adhesion on hydroxyapatite and stainless steel with all three bacteria. (2) Anti-adhesive effect was reduced to 85-90s on glass. Compound 12 (amphoteric). (1) 98-99s antiadhesion with all 3 bacteria on glass. (2) Approximately 50-8048 anti-adhesion with all 3
bacteria on stainless steel and hydroxyapatite. In order for the graft copolymers to be anti-adhesive they must first of all adsorb sufficiently strongly onto a substratum so that they can withstand the washing stage prior to the bacterial adhesion experiments. Thus substratum specificity would be expected as a graft copolymer with a particular backbone structure would adsorb differently on different substrata. It can be seen from Tables 3, 4 and 5, that Compound 12 was the best copolymer on glass, while Compound 11 was the best copolymer on both stainless steel and hydroxyapatite. It is suggested that Compounds l-9 were not effective because most of their backbone is composed of MMA, which does not adsorb strongly enough onto hydrophilic (and charged) substrata. Compounds 11 and 12 can be considered to be bacterially non-specific as they gave similar results with all three bacteria, In contrast, Compound 10 demonstrated both bacterial and substrate specificity. Compound 10 was anti-adhesive on glass (and to a lesser extent on stainless steel) against both Pseudomonas sp. (NCMB 2021) and S.murcescens (NCIB 122il), but not S.mutans (NCTC 10449). This indicates that in this case the charged backbone was having some effect on bacterial adhesion levels. The fact that PDMAEM adsorbed onto glass, decreased S.murcescens (NCIb 12211) and increased S.mutans (NCTC 10449) adhesion levels was additional evidence. However, the surprising observation was that on glass Copolymer 10 was anti-adhesive to Pseudomonas sp. (NCMB 2021), while PDMAEM increased adhesion levels. The objective of the experiments described in this paper was to investigate the feasibility of making hydrophilic substrata anti-adhesive to bacteria by the adsorption of PEG-containing compounds. The intention was to achieve anti-adhesive effects equivalent to those obtained by adsorbing linear ethoxylated surfactants onto hydrophobic substrata [7,8]. This has been achieved. It has been shown that charged graft copolymers can be adsorbed onto hydrophilic surfaces rendering them anti-adhesive to bacteria. It is proposed that the graft copolymers adsorb onto substrata principally by their charged backbone, leaving the
303
PEG chains pointing out into the aqueous phase. This has been confirmed by Nuclear Magnetic Resonance (NMR) studies of Compound 11 adsorbed onto hydroxyapatite (unpublished results). Experiments with PMAA and PDMAEM have shown that the charged polymers do have some effects on bacterial adhesion levels, but the antiadhesive results obtained were not of the same order of magnitude as those achieved with the PEG copolymers. Bacterial adhesion is of major ecological significance in the colonisation of both animate and inanimate surfaces. The attachment of a bacterium at a surface may prevent it from being removed from the system in flowing environments e.g. in the oral cavity, rivers, etc.; or provide enriched nutrient conditions at the solid-liquid interface, especially in nutrient deficient habitats, e.g. aquatic. Once attached, bacteria must be able to initiate growth for their proliferation and colonisation of a surface. Thus colonisation depends upon both adhesion and growth. It is likely that adhesion phenomena dominate early biofilm formation, but the composition of the total mature biofilm that develops will be mainly determined by growth of attached bacteria. However, since initial adhesion by definition is a precursor to biofilm formation, it may be of primary importance in terms of the prevention or control of biofilm formation. We are proposing that preventing, or at least reducing the initial adhesion of bacteria to a surface will result in a significant reduction in subsequent biofilm formation. The experiments described in this paper are only concerned with the initial adhesive stage. Whether the graft copolymers would have any effect on growth and colonisation of adhered bacteria is open to question. We are of the opinion that the copolymers would have no significant effect on the growth and colonisation of adhered bacteria, and that this could only be achieved by toxic effects. The effect of the copolymers on colonisation could be investigated by using a continuous flow-through biofouling rig [8] where surface coverage depends upon both adhesion and resultant growth of bacteria. However, in these types of experiments it would be necessary to monitor the behaviour of the copolymer, since there would be no guarantee
that it would remain adsorbed onto a surface under these increased flow conditions. Theoretically it would also be possible to separate the adhesion from the growth stage in order to determine their relative contributions to the colonisation of a surface. This could be achieved for example by performing an adhesion experiment, and placing the substrata containing attached bacteria under carefully controlled conditions so that nutrient flows directly over the surface in such a way that no additional adhesion could take place. Thus further colonisation of a surface would only be due to growth. This could be monitored under a microscope, and photographs taken at regular periods. The anti-adhesive effect could be considered to be a kinetic phenomenon, slowing down the ‘inevitable’ bacterial adhesion and colonisation of solid surfaces. The rate of reduction of bacterial adhesion and relative importance of adhesion versus growth (dependent amongst other things upon bacterial concentration and nutrient conditions) would determine whether the anti-adhesive effect was of ecological significance. At this stage, we consider the results presented in this paper to be no more than preliminary observations. The concept of an anti-adhesive surface has been proven, but additional synthetic chemistry and more sophisticated testing procedures are required in order to identify further structure-activity relationships and to achieve optimisation of the anti-adhesive effect.
ACKNOWLEDGEMENTS The authors acknowledge invaluable discussions with Mr. J.F. Jaworzyn and Dr. M.A. Eakin, and experimental assistance of Mrs. E.A. Twose.
REFERENCES [l] Ellwood, D.C., Melling, J. and Rutter, P. (Eds.) (1979) Adhesion of Microorganisms to Surfaces. Academic Press, London. [2] Bitton, G. and Marshall, K.C. (Eds.) (1980) Adsorption of Microorganisms to Surfaces. John Wiley and Sons, New York, NY.
304 [3] Mergenhagen, SE. and Rosan, B. (Eds.) (1985) Molecular Basis of Oral Microbial Adhesion. American Society for Microbiology, Washington, DC. [4] Dempsey, M.J. (1981) Marine bacterial fouling: a scanning electron microscope study. Mar. Biol. 61, 305-315. [5] Dempsey, M.J. (1981) Colonisation of anti-fouling paints by marine bacteria. Bot. Mar. 24, 185-191. [6] McCoy, W.F., Bryers, J.D., Robbins, J. and Costerton, J.W. (1981) Observations of fouling biofilm formation. Can. J. Microbial. 27, 910-917. [7] Humphries, M., Jaworzyn, J.F. and Cantwell, J.B. (1986) The effect of a range of biological polymers and synthetic
surfactants on the adhesion of a marine Pseudomonas sp. strain NCMB 2021 to hydrophilic and hydrophobic surfaces. FEMS Microbial. Ecol. 38, 299-308. [8] Humphries, M., Jawoizyn, J.F., Cantwell, J.B. and Eakin, A. (1987) The use of non-ionic ethoxylated and propoxylated surfactants to prevent the adhesion of bacteria to solid surfaces. FEMS Microbial. Lett. 42, 91-101. [9] Verran, J., Drucker, D.B. and Taylor, C.J. (1980) Feasibility of using automatic image analysis for measuring deposition of Streptococcus mutans onto glass in terms of percentage coverage and mean clump size. Microbios. 29, 161-169.
45 (1987) 297-304
297
FEC 00134
The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces Martyn
Humphries,
Jozef
Nemcek,
John
B. Cantwell
and John
Corporate Collard Science Group, ICI PLC, Runcorn, Cheshve,
J. Gerrard
U.K.
Received 24 February 1987 Revision received 2 June 1987 Accepted 9 June 1987
Key words:
Polyethylene
glycol (PEG) graft copolymers;
1. SUMMARY Twelve graft copolymers have been evaluated for their ability to prevent the adhesion of bacteria to substrata. The copolymers had polyethylene glycol (PEG) side-chains (‘teeth’) and a backbone that was either uncharged, acidic, basic or amphoteric. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite substrata, and 2-h petri-dish adhesion experiments performed with bacteria isolated from marine (Pseudomonas sp. NCMB 2021) paper mill (Smarcescens NCIB 12211) and oral (Smutans NCTC 10449) environments. The copolymers containing the most charged groups in the backbone had the most significant effect on bacterial adhesion levels, with anti-adhesive effects up to 99% achieved. An amphoteric copolymer (Compound 12) on glass, and acidic copolymer (Compound 11) on stainless steel and hydroxyapatite gave the most impressive anti-adhesive effects. These copolymers had nonspecific bacterial anti-adhesive properties.
Correspondence to: M. Humphries, Corporate Colloid Science Group, ICI PLC. P.O. Box 11, The Heath, Runcorn, Cheshire WA7 4QE. U.K. 016%6496/87/$03.50
0 1987 Federation
of European
Microbiological
Anti-adhesive
compounds;
Hydrophilic
substrata
It is proposed that the graft copolymers adsorbed onto hydrophilic surfaces via their charged backbone in such a way that the PEG side-chains were pointing out into the aqueous phase, and it was this orientation that was responsible for the observed anti-adhesive effect.
2. INTRODUCTION Bacterial adhesion to solid surfaces [1,2] is a universal and usually unwanted phenomenon which can be considered to be the primary event, for example, in dental plaque formation [3], marine fouling [4,5] and biofilm formation in circulating water systems [6]. Previous papers [7,8] have shown that the adsorption of ethoxylated (and propoxylated) surfactants onto hydrophobic, but not hydrophilic substrata resulted in significant reductions in bacterial adhesion levels in laboratory experiments. This ‘anti-adhesive’ effect was believed to be due to the specific mode of adsorption of linear ethoxylated surfactants on hydrophobic (as opposed to hydrophilic) substrata, i.e. via their hydrophobe in such a way that the polyethylene glycol (PEG) chains were pointing out into the aqueous phase. It was proposed that the hydroSocieties
298
philic, highly hydrated PEG chains provided a steric barrier which prevented the adhesion of bacteria. We suggested that a certain density of PEG chains at the interface would be necessary to achieve the anti-adhesive effect. It was proposed that in theory it should be possible to render any surface anti-adhesive assuming that molecules could be adsorbed and oriented in such a way as to give a high density of PEG chains at the interface. The objective of the experiments described in this paper was to test this hypothesis. The main difficulty was seen to be achieving the adsorption of hydrophilic molecules onto a hydrophilic substratum in an aqueous environment. We believed that this was most likely to occur with polymeric molecules, which having a multiplicity of anchoring points should be more substantive to a surface than the corresponding monomeric compounds. Thus we have synthesised a number of graft copolymers which have a ‘backbone’ which can be uncharged, acidic, basic or amphoteric, with PEG chains as ‘teeth’. The design intention was that the backbone would interact with the substratum in such a way that the PEG chains would point out into the aqueous phase, resulting in an anti-adhesive surface. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite surfaces. Their effects on the adhesion of a marine bacterium (Pseudomonas sp NCMB 2021) paper mill bacterium (S.marcescens NCIB 12211) and an oral bacterium (S.mutans NCTC 10449) were investigated.
3. METHODS 3.1. Synthesis of graft copolymers The graft copolymers (‘combs’) were made by free radical copolymerisation of cu-methoxy-wmethacroyl polyethylene glycols (‘teeth’) with various combinations of methyl methacrylate (MMA)(CH, = C(CH,)CO,CH,), methacrylic acid (MAA) (CH, = C(CH,)CO,H) and N,N-dimethyl-2 aminoethyl methacrylate (DMAEM) (CH, = C(CH,)CO,CH,CH,N(CH,),) in the backbone. 3.1.1. Monomers. Methyl methacrylate (ICI Mond Division), methacrylic acid (BDH) and
IV, N-dimethylaminoethyl methacrylate (Aldrich) were distilled under reduced pressure and stored at 4°C before use. Methoxy polyethyleneglycol methacrylates (for Compounds l-4) were prepared using the general method described below: Freshly distilled methacroyl chloride (0.11 mol) was slowly added to a 30% w/v solution of 2,6-dimethyl pyridine (0.11 moles) [2,6-lutidine (BDH), which was distilled under reduced pressure before use] in toluene, in a 3-necked round bottom flask. The flask was equipped with a dropping funnel, magnetic stirrer, nitrogen bleed and condenser. Some fuming occurred during addition, and a white precipitate formed. The mixture was cooled in an ice bath and methoxy-ended polyethyleneglycol [dried by bubbling nitrogen through the heated liquid (115 “C) under reduced pressure] (0.10 mol, Aldrich) was added dropwise, with stirring, under a nitrogen blanket. A copious white precipitate formed as the reaction proceeded. The addition took 3 h, and on completion the reaction mixture was stirred on ice for an additional 2 h, before the reaction flask was allowed to reach room temperature. The precipitated 2,6-lutidine hydrochloride was filtered off and washed with small amounts of toluene. The combined filtrate was dried down on a rotary evaporator at 45°C under reduced pressure. The product (89-97s yield) was analysed by infra-red, nuclear magnetic resonance and by vinyl bond titration. The methoxy polyethyleneglycol methacrylates were stored at 4 o C before use. Methoxypolyethyleneglycol methacrylate 400 (= nominal M, - Polysciences) was used as obtained for Compounds 5-12. Nuclear Magnetic Resonance spectroscopy revealed the M, to be approximately 420, i.e. the polyethyleneglycol chain had a M, of approximately 350. 3.1.2. Polymerisations. A general method is described below: Free radical polymerisations were performed at 70” C in a 600-ml reaction flask equipped with nitrogen bleed, water cooled condenser, thermometer and stirrer. The reaction vessel was placed in a thermostatically controlled oil bath. Monomers in selected molar ratios (total 25 g) were added together with 300 ml of solvent
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(toluene or industrial methylated spirits/water mixtures) and heated to 70 “C. An aliquot of the initiator (1.8 x 1O-3 mol) 2,2’-azobis (2-methylpropionitrile) (BDH) or 4,4’-azobis (4cyanovaleric acid) (Aldrich) in 20 ml solvent was added and the mixture polymerised with stirring at 200 rpm for 24 h. A second aliquot of initiator (7 X lop4 mol) in 20 ml solvent was then added and the polymerisation continued for a further 24 h. The copolymers were isolated by evaporation under high vacuum of volatile materials (residual starting materials) present in the reaction mixture. The theoretical average compositions of the copolymers were largely confirmed by chemical analysis, nuclear magnetic resonance and acid-base titrations. The average composition of the copolymers are given in Table 1. An example of the structure of a copolymer containing MMA, MAA and DMAEM is given below:
(CH,CH,O),CH, MMA
MA.4
DMAEM
PEG Methacrylate
The M,s of the copolymers were determined by gel permeation chromatography in dimethylformamide-lithium bromide system, using polyethylene oxide as standard. The A4,s were in the range 35 000-70 000 and polydispersities (M,/Mn) 1.5-3 (typical values for free radical polymerisations). Due to the lack of suitable standards the M, values should only be taken as a guideline. 3.1.3. Polymethactylic acid and poly DMAEM. An aqueous solution of polymethacrylic acid (PMMA) of M, 25000 was obtained from Allied Colloids (Versicol-K13). DMAEM Poly (PDMAEM) of M, approximately 30 000 was prepared in our laboratory. 3.2. Organisms and growth conditions A marine Pseudomonas sp. strain National Collection of Marine Bacteria 2021 (NCMB 2021) was stored on Marine Agar plates and grown up in 100 ml batches of Marine Broth (Difco) in a
G24 Environmental Incubator Shaker (New Brunswick) at 18” C, 200 rev/mm for 17 h. The bacteria were diluted 10 times in 2.4% w/v saline for adhesion experiments. The bacterial concentration was approximately 5 X 106. ml-’ (determined by counting in a haemacytometer). Serratia marcescens strain National Collection of Industrial Bacteria 12211 (NCIB 12211) was stored on Nutrient Agar plates and grown up in 100-ml batches of Nutrient Broth (Oxoid) in the incubator shaker at 30°C, 200 rev/min for 17 h. The bacteria were diluted 10 times in distilled water for adhesion experiments. The bacterial concentration was approximately 1 X lo6 f ml-’ (determined by counting in a haemacytometer). Streptococcus mutans strain National Collection of Type Cultures 10449 (NCTC 10449) was stored on Brain Heart Infusion Agar plates and grown up in loo-ml batches of Brain Heart Infusion Broth (Oxoid) in the incubator shaker at 37” C, 200 rev/mm for 17 h. The bacteria were centrifuged for 10 min at 4000 rev/mm, resuspended in modified Ringer’s salts solution (0.54 g. 1-l NaCl, 0.02 g.l-’ KCl, 0.03 g.ll’ CaCl,, 0.75 g.ll’ sodium mercaptoacetate), recentrifuged, resuspended and diluted 10 times in modified Ringer’s salts solution. The bacterial concentration was approximately 1 X lo6 . ml-’ (determined by counting in a haemacytometer). 3.3. Surfaces of attachment Glass microscope slides, stainless steel discs and hydroxyapatite discs were used as substrata for bacterial adhesion experiments. Glass microscope slides were cut into thirds, immersed in methanol for 10 min, rinsed and stored in distilled water before use. Stainless steel discs were made from a 25-mm rod by parting off 2 mm thick sections on a lathe. The lathe marks were removed using silicone carbide paper on a lapping machine, and the discs given a mirror polish using a micro-cloth and 6-pm diamond resuspended in water. The discs were made hydrophilic by immersing in boiling 50% phosphoric acid for 2 min. The discs were thoroughly rinsed and stored in distilled water before use. After an experiment, the discs were repolished and reheated in acid for re-use. Hy-
300
Table
1
Average
composition
Copolymer number
Backbone nature
1 2 3 4 5 6 7 8 9 10 11 12
Acid Acid Base Base Acid Base Amphoteric Acid Uncharged b Base b Acid b Amphoteric
of the graft copolymers Side-chain
Molar ratios a
(‘+f,)
I
PEG 550 PEG2000 PEG 550 PEG2000 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350 PEG 350
4:l 13:l 4:l 1O:l 4:l 4:l 2.7:1 2:l 2.7 : 1 3 :1 3:l 3:l
II (MMA : MAA DMAEM)
:
1O:l:O 1O:l:O 18:O:l 18:O:l 1O:l:O 18:O:l 36:1.8:1 1:l:O All MMA All DMAEM All MAA 0:1.8:1
a Molar ratio I = No. of monomer units in backbone: No. of side-chains (not including the methacrylate component of the side-chains). Molar ratio II = ratio of monomers in the backbone (not including the methacrylate component of the side-chains). b No MMA in the backbone.
droxyapatite discs were made by compressing hydroxyapatite powder (calcium phosphate tribasic [Ca,,(OH),(PO,),] (Aldrich) in a 3-cm evacuable X-ray die (Specac, Orpington, U.K.). The discs were heated for 4 h in a furnace at 1100 o C. Hydroxyapatite discs were re-used after heating for 2 h at 900°C. The discs were equilibrated in distilled water overnight prior to bacterial adhesion experiments. 3.4. Bacterial adhesion experiments Glass microscope slides, stainless steel discs and hydroxyapatite discs were immersed in 1% w/v aqueous solutions of the copolymers (also PMAA and PDMAEM) at ambient temperature for 5 min. The slides and discs were given a standard wash by immersing and shaking 5 times in a container of flowing water, and placed in polystyrene petri-dishes. 30 ml of bacterial suspenof Pseudomonas sp. (NCMB 2021) sion Xmarcescens (NCIB 12211) or Xmutans (NCTC 10449) was added to each petri-dish. After 2 h at ambient temperature, the slides and discs were taken out of the bacterial suspension and loosely
attached bacteria removed by immersing and shaking 5 times in a container of flowing water. Adhered bacteria were stained using Loeffler’s Methylene Blue (0.3 g methylene blue, 30 ml 90% ethanol, 100 ml water). Computerised image analysis (Kontron Image Process System) was used to measure bacterial adhesion levels [9]. Percentage adhesion of copolymer-treated substrate relative to untreated controls was calculated = %antiadhesion (0% anti-adhesion = control, 100% antiadhesion = no adhered bacteria). The experiments were performed twice, and mean values calculated.
4. RESULTS Table 2 gives the percentage surface area of substrata (glass, stainless steel and hydroxyapatite) covered by adhered bacteria (= % adhesion) after 2 h petri-dish adhesion experiments with Pseudomonas sp. (NCMB 2021) Xmarcestens (NCIB 12211) and Smutans (NCTC 10449). Tables 3, 4 and 5 show the effect of adsorbing the graft copolymers onto glass, stainless steel and hydroxyapatite substrata prior to 2 h petri-dish adhesion experiments. The results are expressed as % reduction in bacterial adhesion levels compared to untreated controls (= % anti-adhesion). Mean values of duplicate experiments are given.
5. DISCUSSION
The graft copolymers (Table 1) can be conveniently divided into those which contain MMA in
Table 2 Percentage surface area covered by adhered bacteria after 2 h petri-dish adhesion experiments with Pseudomonas sp. (NCMB 2021), Smarcescens (NCIB 12211) and S.mutans (NCTC 10449) %Adhesion
Glass Stainless steel Hydroxyapatite
Pseudomonas
S.mnrcescens
6.8
9.1
S.mutans 18.1
4.2 3.9
7.7 3.6
28.0 27.9
301 Table 3
Table 5
The effect of adsorbing graft copolymers onto glass, stainless steel and hydroxyapatite surfaces on the adhesion of Pseudomonas sp. (NCMB 2021) during 2 h petri-dish adhesion experiments
The effect of adsorbing graft copolymers onto glass, stainless steel and hydroxyapatite surfaces on the adhesion of Xmutons (NCTC 10449) during 2 h petri-dish adhesion experiments.
Mean value of duplicate experiments and the spread of results is given. Graft copolymer
Glass
PMAA PDMAEM
Hydroxyapatite
Stainless steel
47* 4 +63+10
30+ 32k 35k 1+ 17* 33i 28+ 32* 185 6* 95+ 75*
l+ 8 24k 0 35* 7 8+14 14* 4 37+ 6 21& 4 34+ 2 43* 1 81k 3 96+ 2 81+ 3
+9* 6 14*10 71rfr 4 20+ 1 24+ 8 43+10 52* 7 54* 4 52k 5 97+ 1 85+ 2 98k 1
1 2 3 4 5 6 7 8 9 10 11 12
+
% Anti-adhesion
28* +31* -
1 3 4 9 9 4 2 2 7 5 1 4
70& 2 +95*17 -
7 4
= Increase in bacterial adhesion levels.
Table 4 The effect of adsorbing graft copolymers onto glass, stainless steel and hydroxyapatite surfaces on the adhesion of S.marcescens (NCIB 12211) during 2 h petri-dish adhesion experiments Mean value of duplicate experiments and the spread of results is given. Graft copolymer
% Anti-adhesion Glass
Hydroxyapatite
12*
+10*
I
23k
2 3 4 5 6 7 8
30* 4 74* 0 37* 2 21* 2 49+10 37+ 6 24+ 8
29+10 17* 7 lo+ 2 15+ 1 26kll 3*10 9+ 6
9 10 11 12
43+ 99* 86k 99+
4 1 3 1
lo* 90+ 95* 55*
2 2 1 4
PMAA PDMAEM
17* 75*
4 2
22+ 68?
8 5
+
6
Stainless steel 5
= Increase in bacterial adhesion levels.
5
20+12 8k 5 18k 4 21+ 4 +4* 5 53+ 9 +14*16 52+ 23k 94+ 64k
2 6 2 5
77+ 1 +23+11
Mean value of duplicate experiments and the spread of results is given. Graft copolymer
% Anti-adhesion Stainless steel
Glass
1 2 3 4 5
58+ 15& 75+ 55+ 55+
1 6 4 1 3
11+ +I+13 28+ 11* 6&
6
345
2
+11*11
7 8 9
30* 7 38+10 52+ 4 11* 91+ 98k
10 11 12 PMAA PDMAEM
+
3 1 1
7*11 +38+ 5
17* 24* +15* 17* 94i_ 47*
Hydroxyapatite
6 2 8
165 +2+ +fi* 18k +105
6 I 7 3 9
8k
2
5 7 7
13* 32* 6k
1 8 9
4 4 5
21* 96+ 68k
2 1 4
3
17* 7 +46+12
71+ 2 +34+10
= Increase in bacterial adhesion levels.
the backbone (Compounds l-9) and those which do not (Compounds 10-12). Of the former group, Compounds l-7 contain mostly MMA with only a few acidic (MM) and/or basic (DMAEM) groups in the backbone. Compound 8 contains approximately equal numbers of MMA and acidic groups, while Compound 9 is composed of only MMA, i.e. no charged groups. In contrast, Compounds lo-12 have all acidic and/or basic groups in the backbone. The results in Tables 3, 4 and 5 show that it was the latter group that gave the most impressive anti-adhesive effects. The design intention of the graft copolymers was that the charged backbone would interact with the substratum in such a way that there was a high density of PEG chains pointing out into the aqueous phase, and that this orientation was responsible for the anti-adhesive effect, i.e. we have somewhat simplistically divided the copolymers into an effect end (PEG side-chains) and adsorbing end (backbone). However, the charged groups in the backbone of the graft copolymers could also be a factor in bacterial adhesion. For this reason, the effect of a standard acidic and basic polymer
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was investigated. Polymethacrylic acid (PMAA) and poly-N, N-dimethylaminoethyl methacrylate (PDMAEM) were chosen. They could be considered to be Compounds 10 and 11, respectively, without the PEG chains. Results in Tables 3, 4 and 5 show that PMAA and PDMAEM do have some effect on bacterial adhesion levels. PMAA gave approximately 70% anti-adhesive effect with all 3 bacteria on hydroxyapatite, but little effect on glass and stainless steel. PDMAEM actually increased bacterial adhesion levels with Pseudomonas sp. (NCMB 2021) and S.mutans (NCTC 10449) on all 3 surfaces, and with Smarcescens (NCIB 12211) on hydroxyapatite. However, PDMAEM gave approximately 70% anti-adhesive effect with Xmarcescens (NCIB 12211) on glass and stainless steel. Thus for the PEG chains of the graft copolymers to be having a significant role in preventing bacterial adhesion, it was considered necessary to obtain anti-adhesive effects well in excess of the 70% level. Anti-adhesive levels of 90-95s or greater were somewhat arbitrarily considered to be significant. Tables 3, 4 and 5 show that of Compounds 1-9, only Compound 6 yields anti-adhesive effects at the 70% level, and this was only achieved on glass. These anti-adhesive effects were not considered to be significant. In contrast, Compounds 10, 11 and 12 gave anti-adhesive effects both within and above the 90-95% level, even as high as 99% anti-adhesion. The results in Tables 3, 4 and 5 demonstrate both bacterial and substratum specificity. The major observations are listed below. Compound 10 (basic). (1) No significant antiadhesive effect with all 3 bacteria on hydroxyapatite, or with S.mutans (NCTC 10449) on all 3 surfaces. (2) Yields greater than 95% anti-adhesion with Pseudomonas sp. (NCMB 2021) and S.murcescens (NCIB 12211) on glass. This was reduced to 80-90s anti-adhesion on stainless steel. Compound II (acidic). (1) Approximately 95% anti-adhesion on hydroxyapatite and stainless steel with all three bacteria. (2) Anti-adhesive effect was reduced to 85-90s on glass. Compound 12 (amphoteric). (1) 98-99s antiadhesion with all 3 bacteria on glass. (2) Approximately 50-8048 anti-adhesion with all 3
bacteria on stainless steel and hydroxyapatite. In order for the graft copolymers to be anti-adhesive they must first of all adsorb sufficiently strongly onto a substratum so that they can withstand the washing stage prior to the bacterial adhesion experiments. Thus substratum specificity would be expected as a graft copolymer with a particular backbone structure would adsorb differently on different substrata. It can be seen from Tables 3, 4 and 5, that Compound 12 was the best copolymer on glass, while Compound 11 was the best copolymer on both stainless steel and hydroxyapatite. It is suggested that Compounds l-9 were not effective because most of their backbone is composed of MMA, which does not adsorb strongly enough onto hydrophilic (and charged) substrata. Compounds 11 and 12 can be considered to be bacterially non-specific as they gave similar results with all three bacteria, In contrast, Compound 10 demonstrated both bacterial and substrate specificity. Compound 10 was anti-adhesive on glass (and to a lesser extent on stainless steel) against both Pseudomonas sp. (NCMB 2021) and S.murcescens (NCIB 122il), but not S.mutans (NCTC 10449). This indicates that in this case the charged backbone was having some effect on bacterial adhesion levels. The fact that PDMAEM adsorbed onto glass, decreased S.murcescens (NCIb 12211) and increased S.mutans (NCTC 10449) adhesion levels was additional evidence. However, the surprising observation was that on glass Copolymer 10 was anti-adhesive to Pseudomonas sp. (NCMB 2021), while PDMAEM increased adhesion levels. The objective of the experiments described in this paper was to investigate the feasibility of making hydrophilic substrata anti-adhesive to bacteria by the adsorption of PEG-containing compounds. The intention was to achieve anti-adhesive effects equivalent to those obtained by adsorbing linear ethoxylated surfactants onto hydrophobic substrata [7,8]. This has been achieved. It has been shown that charged graft copolymers can be adsorbed onto hydrophilic surfaces rendering them anti-adhesive to bacteria. It is proposed that the graft copolymers adsorb onto substrata principally by their charged backbone, leaving the
303
PEG chains pointing out into the aqueous phase. This has been confirmed by Nuclear Magnetic Resonance (NMR) studies of Compound 11 adsorbed onto hydroxyapatite (unpublished results). Experiments with PMAA and PDMAEM have shown that the charged polymers do have some effects on bacterial adhesion levels, but the antiadhesive results obtained were not of the same order of magnitude as those achieved with the PEG copolymers. Bacterial adhesion is of major ecological significance in the colonisation of both animate and inanimate surfaces. The attachment of a bacterium at a surface may prevent it from being removed from the system in flowing environments e.g. in the oral cavity, rivers, etc.; or provide enriched nutrient conditions at the solid-liquid interface, especially in nutrient deficient habitats, e.g. aquatic. Once attached, bacteria must be able to initiate growth for their proliferation and colonisation of a surface. Thus colonisation depends upon both adhesion and growth. It is likely that adhesion phenomena dominate early biofilm formation, but the composition of the total mature biofilm that develops will be mainly determined by growth of attached bacteria. However, since initial adhesion by definition is a precursor to biofilm formation, it may be of primary importance in terms of the prevention or control of biofilm formation. We are proposing that preventing, or at least reducing the initial adhesion of bacteria to a surface will result in a significant reduction in subsequent biofilm formation. The experiments described in this paper are only concerned with the initial adhesive stage. Whether the graft copolymers would have any effect on growth and colonisation of adhered bacteria is open to question. We are of the opinion that the copolymers would have no significant effect on the growth and colonisation of adhered bacteria, and that this could only be achieved by toxic effects. The effect of the copolymers on colonisation could be investigated by using a continuous flow-through biofouling rig [8] where surface coverage depends upon both adhesion and resultant growth of bacteria. However, in these types of experiments it would be necessary to monitor the behaviour of the copolymer, since there would be no guarantee
that it would remain adsorbed onto a surface under these increased flow conditions. Theoretically it would also be possible to separate the adhesion from the growth stage in order to determine their relative contributions to the colonisation of a surface. This could be achieved for example by performing an adhesion experiment, and placing the substrata containing attached bacteria under carefully controlled conditions so that nutrient flows directly over the surface in such a way that no additional adhesion could take place. Thus further colonisation of a surface would only be due to growth. This could be monitored under a microscope, and photographs taken at regular periods. The anti-adhesive effect could be considered to be a kinetic phenomenon, slowing down the ‘inevitable’ bacterial adhesion and colonisation of solid surfaces. The rate of reduction of bacterial adhesion and relative importance of adhesion versus growth (dependent amongst other things upon bacterial concentration and nutrient conditions) would determine whether the anti-adhesive effect was of ecological significance. At this stage, we consider the results presented in this paper to be no more than preliminary observations. The concept of an anti-adhesive surface has been proven, but additional synthetic chemistry and more sophisticated testing procedures are required in order to identify further structure-activity relationships and to achieve optimisation of the anti-adhesive effect.
ACKNOWLEDGEMENTS The authors acknowledge invaluable discussions with Mr. J.F. Jaworzyn and Dr. M.A. Eakin, and experimental assistance of Mrs. E.A. Twose.
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304 [3] Mergenhagen, SE. and Rosan, B. (Eds.) (1985) Molecular Basis of Oral Microbial Adhesion. American Society for Microbiology, Washington, DC. [4] Dempsey, M.J. (1981) Marine bacterial fouling: a scanning electron microscope study. Mar. Biol. 61, 305-315. [5] Dempsey, M.J. (1981) Colonisation of anti-fouling paints by marine bacteria. Bot. Mar. 24, 185-191. [6] McCoy, W.F., Bryers, J.D., Robbins, J. and Costerton, J.W. (1981) Observations of fouling biofilm formation. Can. J. Microbial. 27, 910-917. [7] Humphries, M., Jaworzyn, J.F. and Cantwell, J.B. (1986) The effect of a range of biological polymers and synthetic
surfactants on the adhesion of a marine Pseudomonas sp. strain NCMB 2021 to hydrophilic and hydrophobic surfaces. FEMS Microbial. Ecol. 38, 299-308. [8] Humphries, M., Jawoizyn, J.F., Cantwell, J.B. and Eakin, A. (1987) The use of non-ionic ethoxylated and propoxylated surfactants to prevent the adhesion of bacteria to solid surfaces. FEMS Microbial. Lett. 42, 91-101. [9] Verran, J., Drucker, D.B. and Taylor, C.J. (1980) Feasibility of using automatic image analysis for measuring deposition of Streptococcus mutans onto glass in terms of percentage coverage and mean clump size. Microbios. 29, 161-169.