APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2050-2055 Vol. 53, No. 90099-2240/87/092050-06$02.00/0Copyright © 1987, American Society for Microbiology

Antimicrobial Activity of Tertiary Amine Covalently Bonded to aPolystyrene Fiber

YOSHIHIRO ENDO,* TOHRU TANI, AND MASASHI KODAMA

Department of Surgery, Shiga University of Medical Science, Seta Tsukinowa, Otsu-shi, Shiga 520-21, Japan

Received 2 February 1987/Accepted 4 June 1987

Tertiary amine was covalently bonded to a polystyrene fiber and examined for antibacterial activity. Thetertiary amine covalently bonded to a polystyrene fiber (TAF) showed a high antimicrobial activity againstEscherichia coli. TAF exhibited a stronger antibacterial activity against gram-negative bacteria (E. coli,Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens) thanagainst gram-positive bacteria (Staphylococcus aureus and Streptococcus faecalis) or Candida albicans. Thisactivity against E. coli was accentuated by 0.1% deoxycholate or 10 mg of actinomycin D per ml, to which E.coli is normally not susceptible. This implies that TAF causes an increase of the bacterial outer membranepermeability. On the other hand, the antimicrobial activity was inhibited by adding Mg2+ or by lowering thepH. This suggests an electrostatic interaction between the bacterial cell wall and TAF. Scanning electronmicroscopy showed that E. coli cells were initially attached to TAF, with many projections on the cell surface,but then were apparently lysed after contact for 4 h. Taken together, these results imply that bacteria initiallyinteract with TAF by an electrostatic force between the anionic bacterial outer membrane and the cationictertiary amine residues of TAF and that longer contact with TAF damages the bacterial outer membranestructure and increases its permeability.

Bacterial infection is still a major problem in patients whohave artificial organs such as a vascular graft or a continuousambulatory peritoneal dialysis tube and those who aretreated by using blood-perfused artificial organs. In treatingpatients with hepatic disorders, it is especially important toprotect against bacterial infection, which is a major factor inthe occurrence of multiple organ failure. For that reason,when hemoperfusion is performed on a patient with jaundiceand bacteremia, it is important for the bilirubin adsorbent tohave an antimicrobial activity.A number of attempts have been made to attach an

antimicrobial agent such as an antibiotic (6, 9, 12), quater-nary ammonium compound (3, 8, 27), hexachlorophene (2),or iodide (26) to an insoluble matrix. These products wereintended to be used as water disinfectants, as antimicrobialsurfaces, or for investigations of the mechanism of action ofdrugs. However, there have been no reports of a materialwhich safely exhibits an antimicrobial activity in blood.

Previously we reported that polymyxin B immobilized onfiber (5), which could be safely used and which detoxifiedendotoxin in the blood, had an antimicrobial activity againstgram-negative bacteria (4). In this study we experimentedwith a simpler model of insoluble antimicrobial compoundsthan polymyxin B immobilized on fiber.

Tertiary amine covalently bonded to polystyrene fiber(TAF) was originally developed as an adsorbent for use inthe blood (7). Direct hemoperfusion or plasma perfusionwith TAF has been applied to remove bilirubin from patientswith hyperbilirubinemia or hepatic failure.Many insolubilized quaternary ammonium salts have been

shown to have an antimicrobial activity (3, 8, 27), but no onehas reported on the antimicrobial activity of insolubilizedtertiary amino compounds. We have found that TAF has anantibacterial effect and have sought to determine the mech-

* Corresponding author.

anism of its antibacterial action. In this study we describe itsantimicrobial character. The results suggest that TAF dam-ages the bacterial outer membrane.

MATERIALS AND METHODS

Antibacterial fiber. Tertiary amine was covalently bondedto polystyrene fiber. This poly(N,N-dimethylaminomethyl-styrene) (TAF) was prepared by M. Murakami and K.Teramoto, Toray Fibers and Textiles Research Laborato-ries, Otsu, Shiga, Japan (Fig. 1). They reported that 1 g ofTAF contained 2.44 mmol of covalently bound tertiaryamine. This fiber was soaked in distilled water and wassterilized and made hydrophilic by being autoclaved at 121°Cfor 15 min.

Microorganisms and media. The major microorganismused in this experiment was Escherichia coli ATCC 25922.Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumo-niae ATCC 27736, Salmonella typhimurium ATCC 13311,Serratia marcescens ATCC 8100, Staphylococcus aureusATCC 25923, and Streptococcus faecalis ATCC 29212 wereused for the antimicrobial spectrum. Candida albicans wasprovided by K. Tatewaki, Department of Laboratory Med-icine, Shiga University of Medical Science. Bacteria wereinoculated on Trypto-Soya agar plates (TSA plates; NissuiPharmaceutical Co. Ltd., Tokyo, Japan) or blood agar plates(Nissui) and grown overnight at 37°C. C. albicans wasinoculated on Sabouraud dextrose agar plates (Nissui) andgrown at 25°C for 3 days. Each culture was harvested andwashed twice in 0.05 M Tris hydrochloride (Nakarai Chem-icals, Ltd., Kyoto, Japan) buffer (pH 7.6) or phosphate-buffered saline (pH 7.4) (Dulbecco PBS (-); Nissui) bycentrifugation at 1,600 x g for 10 min at room temperature.Washed cell suspensions were resuspended in the samebuffer as described above and then diluted to the desired

2050

on April 6, 2021 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

ANTIMICROBIAL ACTIVITY OF TERTIARY AMINE FIBER

CH2CH i-n

S NCH2N

APPL. ENVIRON. MICROBIOL.

7 I Icontrol (TSA. DHL agar)

6

0

C 1>~ 3 TAF (TSA)

0o3 \1J \ TAF (DHL agar)

O *_g** .*ui0 1 2 3 4

HoursFIG. 3. Viability of TAF-treated E. coli cells on TSA and sus-

ceptibility of TAF-treated E. coli cells to DHL agar. The number ofviable E. coli cells decreased after E. coli had been shaken withTAF. TAF-treated E. coli was inhibited on DHL agar, althoughnative E. coli was not susceptible to DHL agar. Sublethal damage ofTAF-treated E. coli was observed on DHL agar. The asteriskindicates the time at which no survivors were detected.

coli. After being shaken with TAF, E. coli was incubated at37°C for 30 min with or without 10 mg of actinomycin D(Banyu Pharmaceutical Co., Ltd., Tokyo, Japan), a natu-rally impermeant hydrophobic drug, per ml. Without TAF,E. coli was not susceptible to actinomycin D (19). Thereduction in the number of viable cells by TAF was signifi-cantly (P < 0.05) accentuated by actinomycin D, and noviable E. coli cells could be detected after incubation withactinomycin D after only 5 min of contact with TAF. That is,E. coli cells treated with TAF became susceptible to actino-mycin D (Fig. 4).

Inhibitory effect of Mg2+. The E. coli suspension in 0.05 MTris hydrochloride (pH 7.6) was prepared with or without 4or 40 mM Mg2+ to examine the effect of divalent cations onthe antibacterial activity of TAF. The number of viable cellswas counted on DHL agar plates. The number of surviving

TABLE 2. Microbicidal activity of TAF and susceptibility ofTAF-treated gram-negative bacteria to DHL agara

Viability (%) Susceptibility' toOrganism(s) after: DHL agar (%) after:

10 min 1 h 10 min 1 h

Gram-negative bacteriaE. coli 15 0.007 0.015 0.0046P. aeruginosa 0.67 0.0017 0.023 0.002K. pneumoniae 1.4 0.42 0.064 0.003Salmonella typhimurium 66 1.2 0.0017 0.00023Serratia marcescens 1.7 0.018 0.0063 0.00038

Gram-positive bacteriaStaphylococcus aureus 36 20 NDC NDStreptococcus faecalis 22 6.3 ND ND

C. albicans 19 48 ND NDa All experiments except those for susceptibility were carried out at least

twice. The values shown represent the mean of two or more samples.b Susceptibility is defined as follows: (TAF-treated viable cells on

DHL/viable cells on DHL without TAF treatment) x 100.c ND, Not done.

7 . . .

control6

E 4' control (Act. D)

=5 0%,

C)

54TAF

CO)"-O30

2TAF (Act. D)

1 A~-A^ _____,, *0 30 60

Minutes

FIG. 4. Increased outer membrane permeability of TAF-treatedE. coli evaluated by actinomycin D. TAF-treated E. coli wassusceptible to actinomycin D, which normally could not enter thecell. The asterisk indicates the times at which no survivors weredetected.

cells increased with increasing concentrations of Mg2+ (Fig.5). At 30 min the number of surviving E. coli cells treatedwith TAF but without Mg2+ was significantly (P < 0.05)lower than the number of cells with 4 or 40 mM Mg2+. Lowconcentrations of Mg2' dose dependently inhibited the an-tibacterial action of TAF.

Inhibitory effect of lowering pH. The antibacterial activityof TAF was tested in 0.05 M Tris hydrochloride solution atvarious pHs ranging from 4.3 to 7.7 by measuring thenumber of viable TAF-treated E. coli on TSA after shaking.This number was significantly (P < 0.05) decreased fromcontrol values at pHs varying from 4.3 to 7.7, and thereduction in the number of viable E. coli cells by TAF was

7

- 6

= 50

cm4

;3p

*CO 200

~1

00 10 20 30

MinutesFIG. 5. Effect of Mg2+ on the antimicrobial action of TAF.

TAF-treated E. coli ( ) decreased with a decreasing concentra-tion of Mg2+, although E. coli without TAF (------) was unaffected by0, 4, and 40 mM Mg2+. The asterisk indicates the time at which nosurvivors were detected.

2052 ENDO ET AL.

on April 6, 2021 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

ANTIMICROBIAL ACTIVITY OF TERTIARY AMINE FIBER

8

_- - 6o67 control 10, 30 min

06

E 5 *TA10 minco

0 TAF 30 min-J3

24 5 6 7 8

pH

FIG. 6. Effect of pH on the antimicrobial action of TAF. Thenumber of viable TAF-treated E. coli cells decreased with increasingpH, although the number of viable cells in the control sample wasunchanged at the pHs examined.

significantly (P < 0.05) inhibited between pH 4.3 and 5.7 andat pH 7.7 (Fig. 6).No viable cells on TAF in broth. After shaking for 4 or 8 h,

fibers were removed from the tube and put in a heart infusionbroth. However, no bacterial growth was observed in anysample even after 48 h.SEM. The surface of TAF was observed by SEM at an E.

coli suspension of 106 or 109 CFU/ml. E. coli cells wereobserved on the surface of TAF which had been soaked inthe E. coli suspension at 109 CFU/ml for only 10 s. Some ofthem showed numerous projections extending from the cellsurface (Fig. 7A and B). However, E. coli was not observedafter TAF had been shaken with the E. coli suspension at 106CFU/ml for 4 h (Fig. 7C).

DISCUSSION

Tertiary amine was covalently bonded to polystyrenefibers and examined for antibacterial activity. To correctlyevaluate the antimicrobial activity of these fibers, we at-tempted to shake them vigorously with the bacterial suspen-sion by modifying the classical shake flask method (20) toincrease the probability of cell-to-material surface contact.This procedure gave relatively constant values for the anti-microbial activity of materials examined. There are manyreports on the antimicrobial activity of soluble and insolublequaternary ammonium compounds (3, 8, 27), but none oninsoluble tertiary amine compounds. We found that TAFshowed a high antimicrobial activity against E. coli.No release of antimicrobial substances from TAF has been

observed, although there have been reports that the activityof some insoluble antimicrobial agents was due to the releaseof antibacterial substances (9, 26). TAF was developed asone of the adsorbents that could be safely used in the blood(7).Because the arm to which tertiary amine was attached was

too short (less than 0.1 nm) to enter the bacterial cell wall,TAF could directly affect the bacterial cell membrane butprobably not the intracellular organelles. Therefore theantibacterial mechanism of TAF is expected to be similar tothat of the membrane-acting antibiotics such as polymyxinB.

The antimicrobial activity of TAF was specifically effec-tive against gram-negative bacteria. Divalent cations such asMg2+, Ca2 , and Fe2+were reported to maintain the normalpermeability barrier of E. coli against polymyxin B (17, 18,23) and other antibiotics (14, 15). The antibacterial activity ofTAF was also dose dependently inhibited by Mg2+ andincreased with decreasing pH. These results suggest that theelectrostatic interaction is related to the antibacterial activityof TAF. Because divalent cations normally function ascationic bridges between adjacent phosphates of membranelipids in gram-negative bacteria (23), cationic tertiary amineresidues of TAF were suggested to compete with cations atthe active site in the outer membrane. Therefore electro-static interaction between the positively charged TAF andthe negatively charged bacterial surface is suggested.To demonstrate the alteration of outer membrane perme-

ability, we used two hydrophobic agents, actinomycin D anddeoxycholate. The outer membrane forms an impenetrablebarrier against hydrophobic agents (12). Actinomycin D is anormally impermeant hydrophobic drug (19), but its activityagainst gram-negative bacteria is accentuated by outer mem-brane-damaging drugs such as EDTA, colistin, and poly-myxin B (13, 16). The alterations of the outer membrane canbe demonstrated by determining the bacterial susceptibilityto actinomycin D (1, 28). Deoxycholate is one of the deter-gents to which E. coli is normally resistant; it was reportedthat antibiotic-resistant Proteus mirabilis organisms werekilled on MacConkey agar in which the active fraction wasdeoxycholate (24). It has also been used to show sublethaldamage of the outer membrane (11). In this study the actionof TAF against E. coli was also accentuated by using thesetwo agents, suggesting the perturbation of the outer mem-brane by TAF.We observed that the viable-cell count was reduced by

TAF. This result could be explained by two hypotheses,namely that TAF adsorbed only viable E. coli cells or thatTAF killed E. coli. We examined these hypotheses by SEMand incubation in broth. We thought that if TAF adsorbedthe viable bacteria, bacterial growth should be observedfrom TAF in the broth, but no bacterial growth from TAFwhich had had contact with E. coli was observed. However,E. coli was observed on the surface of TAF in the suspensionat 109 CFU/ml at 10 s, and numerous projections or blebsextended from the outside surfaces of the cells. Similarobservations have been reported as morphological evidenceof outer membrane damage by membrane-acting antibioticssuch as polymyxin B and related peptide antibiotics (10, 21,22, 25). Since after being shaken for 4 h at 106 CFU/ml theviable E. coli cells apparently lysed and were not seen onTAF by SEM, it seems that when the bacterial concentrationis relatively high or the contact time is relatively short, theattachment of E. coli on TAF is observed by SEM, althoughin other situations in which the contact between bacteria andTAF is sufficient for outer membrane damage to occur, it isfollowed by prompt bacteriolysis. Therefore the action ofTAF is thought to be not only adsorptive but mainly bacte-ricidal.TAF was examined for the interaction of artificial materi-

als and bacterial cell membranes. It was specifically effectiveagainst gram-negative bacteria. The mechanism of the anti-bacterial action of TAF against E. coli and other susceptiblegram-negative bacteria may be tentatively explained asfollows. Initially E. coli cells interact with TAF by theelectrostatic force between the anionic bacterial outer mem-brane and the cationic tertiary amine residues of TAF, andthis interaction is dose dependently inhibited by divalent

VOL. 53, 1987 2053

on April 6, 2021 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

2054 ENDO ET AL.

cations such as the magnesium ion that normally function ascationic bridges between adjacent phosphates. After contactwith TAF, the outer membrane function as a permeabilitybarrier is damaged, as proved by the susceptibility of TAF-

FIG. 7. SEM of TAF-treated E. coli and the surface of TAF. (A)After 10 s of contact of TAF with E. coli (109 CFU/ml), E. coli cellswere attached on the surface of TAF (magnification x 10,000). (B)Some of the cells showed numerous projections extending from thesurface (magnification, x 20,000). (C) After 4 h E. coli (106 CFU/ml)was not observed (magnification, x 10,000). Arrows represent 0.5,um.

treated E. coli to actinomycin D and deoxycholate, and theouter membrane structure is also damaged, as observed bySEM. This functional and structural damage of the outermembrane causes bacterial death.

APPL. ENVIRON. MICROBIOL.

on April 6, 2021 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

ANTIMICROBIAL ACTIVITY OF TERTIARY AMINE FIBER

ACKNOWLEDGMENTS

We thank K. Tatewaki, Department of Laboratory Medicine,Shiga University of Medical Science, for his advice and for prepa-ration of bacteria. We also thank M. Murakami and K. Teramoto fortheir helpful discussions and for the preparation of antimicrobialfibers.

LITERATURE CITED1. Beckerdite, S., C. Mooney, J. Weiss, R. Franson, and P. Elsbach.

1974. Early and discrete changes in permeability of Escherichiacoli and certain other gram-negative bacteria during killing bygranulocytes. J. Exp. Med. 140:396-409.

2. Davis, A. E., Jr. 1969. Antibacterial plastics, J. Am. Assoc.Contam. Control 1:63-68.

3. Domagk, G. 1935. Eine neue Klasse von Desinfektionsmitteln.Dtsch. Med. Wochenschr. 61:829-832.

4. Endo, Y., K. Hanasawa, T. Tani, T. Oka, T. Yoshioka, and M.Kodama. 1985. The antimicrobial activity and the endotoxindetoxifying activities of polymyxin B immobilized fiber, p.257-258. In J. Ishigami (ed.), Recent advances in chemother-apy. University of Tokyo Press, Tokyo.

5. Hanasawa, K., T. Tani, T. Oka, T. Yoshioka, Y. Endo, M.Horisawa, Y. Nakane, M. Kodama, K. Teramoto, and S.Nishiumi. 1984. A new treatment for endotoxemia with directhemoperfusion by polymyxin immobilized fiber, p. 167-170. InY. Nose, P. S. Malchensky, and J. W. Smith (ed.), Therapeuticapheresis: a critical look. ISAO Press, Cleveland.

6. Harvey, R. A., and R. S. Greco. 1981. The noncovalent bondingof antibiotics to a polytetrafluoroethylene-benzalkonium graft.Ann. Surg. 194:642-647.

7. Idezuki, Y., M. Hamaguchi, S. Hamabe, H. Moriya, T.Nagashima, H. Watanabe, T. Sonoda, K. Teramoto, T. Kikuchi,and H. Tanzawa. 1981. Removal of bilirubin and bile acid with anew anion exchange resin. Trans. Am. Soc. Artif. Intern.Organs 27:428-433.

8. Isquith, A. J., E. A. Abbott, and P. A. Walters. 1972. Surface-bonded antimicrobial activity of an organosilicon quaternaryammonium chloride. Appl. Microbiol. 24:859-863.

9. Kennedy, J. F., and H. C. Tun. 1973. Active insolubilizedantibiotics based on cellulose and cellulose carbonate. Antimi-crob. Agents Chemother. 3:575-579.

10. Koike, M., K. Iida, and T. Matsuo. 1969. Electron microscopicstudies on mode of action of polymyxin B. J. Bacteriol. 97:448-452.

11. Laforce, F. M., and D. S. Boose. 1981. Sublethal damage ofEscherichia coli by lung lavage. Am. Rev. Respir. Dis. 124:733-737.

12. LaPorte, D. C., K. S. Rosenthal, and D. R. Storm. 1977.Inhibition of Escherichia coli growth and respiration by poly-

myxin B covalently attached to agarose beads. Biochemistry16:1642-1648.

13. Leive, L. 1968. Studies on the permeability change produced incoliform bacteria by ethylenediaminetetraacetate. J. Biol.Chem. 243:2373-2380.

14. Muschel, L. H., and J. E. Jackson. 1966. Reversal of thebactericidal action of serum by magnesium ion. J. Bacteriol.91:1399-1402.

15. Nakajima, K. 1967. Structure-activity relationship of colistins.Chem. Pharm. Bull. 15:1219-1224.

16. Nakajima, K., and J. Kawamata. 1965. Effect of colistin on theactinomycin sensitivity of Escherichia coli. Biken J. 8:115-118.

17. Newton, B. A. 1953. Reversal of the antibacterial activity ofpolymyxin by divalent cations. Nature (London) 172:160-161.

18. Newton, B. A. 1956. The properties and mode of action of thepolymyxins. Bacteriol. Rev. 20:14-27.

19. Nikaido, H., and T. Nakae. 1979. The outer membrane ofgram-negative bacteria. Adv. Microb. Physiol. 20:163-250.

20. Purcell, W. P., G. E. Bass, and J. M. Clayton. 1973. Experimen-tal determination of partition coefficients, p. 126-182. In W. P.Purcell (ed.), Strategy of drug design: a guide to biologicalactivity. John Wiley & Sons, Inc., New York.

21. Rosenthal, K. S., P. E. Swanson, and D. R. Storm. 1976.Disruption of Escherichia coli outer membranes by EM 49. Anew membrane active peptide antibiotic. Biochemistry 15:5783-5792.

22. Schindler, P. G., and M. Teuber. 1975. Action of polymyxin Bon bacterial membranes: morphological changes in the cyto-plasm and in the outer membrane of Salmonella typhimuriumand Escherichia coli B. Antimicrob. Agents Chemother. 8:95-104.

23. Storm, D. R., K. S. Rosenthal, and P. E. Swanson. 1977.Polymyxin and related peptide antibiotics. Annu. Rev. Bio-chem. 46:723-763.

24. Sud, I. J., and D. S. Feingold. 1972. Effect of polymyxin B onantibiotic-resistant Proteus mirabilis. Antimicrob. Agents Che-mother. 1:417-421.

25. Suganuma, A., K. Hara, T. Kishida, K. Nakajima, and J.Kawamata. 1968. Cytological changes of Escherichia colicaused by polymyxin E. Biken J. 11:149-155.

26. Taylor, S. L., L. R. Fina, and J. L. Lambert. 1970. New waterdisinfectant: an insoluble quaternary ammonium resin-triiodidecombination that releases bactericide on demand. Appl. Micro-biol. 20:720-722.

27. Walters, P. A., E. A. Abbott, and A. J. Isquith. 1973. Algicidalactivity of a surface-bonded organosilicon quaternary ammo-nium chloride. Appl. Microbiol. 25:253-256.

28. Weiss, J., M. Victor, and P. Elsbach. 1983. Role of charge andhydrophobic interactions in the action of the bactericidal/perme-ability-increasing protein of neutrophils on gram-negative bac-teria. J. Clin. Invest. 71:540-549.

2055VOL. 53, 1987

on April 6, 2021 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/