Vol. 43, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1982, p. 1041-10500099-2240/82/051041-10$02.00/0

Antimicrobial Characteristic of Insoluble AlkylpyridiniumIodide

YOSHIHIRO NAKAGAWA,l* YASUHIKO YAMANO,' TAKAHIKO TAWARATANI,1 HIROKIKOURAI,2 TOKUNARU HORIE,2 AND ISAO SHIBASAKI'

Department of Fermentation Technology, Faculty of Engineering, Osaka University, 2-1, Yamadaoka, Suita-shi, Osaka 565,' and Department ofApplied Chemistry, Technical College of Tokushima University, Minami

josanjima-cho, Tokushima 770,2 Japan

Received 11 September 1981/Accepted 31 December 1981

Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized. Theinsoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers.The insoluble agent [C12(50)] that contained 50% divinylbenzene and had a C12alkyl chain was selected as the most suitable insoluble agent. C12(50) showed poor

durability of the antibacterial activity, but C12(50), which had lost the activity,was refreshed by washing with ethanol. This washing became ineffective after a

few cycles of antibacterial treatment and refreshment. Such C12(50) recovered theactivity upon 1.0 N NaOH treatment. The antibacterial activity of C 12(50)depended on its surface area. It showed high antimicrobial activity against gram-

positive bacteria and also showed activity against gram-negative bacteria andyeasts. But the activities of C12(50) and laurylpyridinium iodide solution were

different against some microbes. The antibacterial activities of the agents were

investigated against Escherichia coli and Micrococcus luteus under variousconditions. The activity of C12(50) was higher at a higher temperature or at a lowercell concentration. The activity of C12(50) decreased on addition of NaCl, glucose,or bovine albumin to the cell suspension or in 0.01 M sodium-potassiumphosphate buffer. C12(50) showed less activity when cells were mixed with deadcells or the supernatant of dead cells killed in an autoclave. The mode of action ofthe laurylpyridinium iodide solution against E. coli and M. Iuteus was similar tothat of C12(50) except for the influence of E. coli cell concentration.

Prevention of microbial contamination offoodstuffs and packaging materials is very im-portant for improving preservation of productsand for preventing qualitative deterioration. Forthis purpose, heating, addition of antimicrobialagents, UV or gamma irradiation, and othermethods are carried out. The addition of antimi-crobial agents consumes less energy than othermethods, but the toxicity of residual agents mustbe taken into consideration.With the use of antimicrobial agents in liquid

or gas, the problem of residues cannot be avoid-ed, but this problem will be solved if the antimi-crobial agents can be insolubilized. Moreover, itis possible to sterilize liquids continuously on alarge scale and to use agents which are prohibit-ed for use in foods because of their strongtoxicities.

Antibiotics (8, 9), organosilicon quaternaryammonium chloride (3, 18), and hexachloro-phene (16) were insolubilized on several materi-als to inhibit microbial growth. Iodide (2, 6, 7,14, 15, 17), silver (5), polymyxin B (11), andquaternary ammonium chloride (4) were alsoinsolubilized to sterilize the microbes. We inves-

tigated the insolubilization of quaternary ammo-nium salts on cation-exchange resins (12, 13) andfound high sterilizing activity. Unfortunately,the agent was released from the complex duringthe sterilization procedure at a very low concen-tration.

In this work, we attempted the insolubiliza-tion of quaternary ammonium salts (alkylpyri-dinium iodides [APyIs]) by covalent binding,selected the most suitable insoluble agent, andinvestigated the durability and recovery of theactivity. Furthermore, the modes of action ofthe insoluble agent and its soluble form wereinvestigated under various conditions.

MATERIALS AND METHODS

Synthesis of insoluble and soluble APyI. A total of 300g of pure 4-vinylpyridine (VP) and divinylbenzene(DVB) was suspended in 3,000 ml of aqueous 3%polyvinyl alcohol solution at a definite moler ratio (VP/DBV = 9:1, 7:3, 1:1, or 3:7). After the suspension wasstirred at room temperature for 60 min, ot,a'-azobis-isobutyronitrile (5 g) was added, and then the mixturewas heated at 90°C for 8 h under vigorous stirring. TheVP-DVB copolymer formed was collected, washed

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1042 NAKAGAWA ET AL.

TABLE 1. Composition of insoluble

Alkyl DVB VP VAPIbchain (mol%) (mol%) mol% mmolUgC8 50.0 25.7 24.3 1.38Clo 50.0 26.6 23.4 1.30C12 10.0 18.9 71.1 2.24

30.0 26.6 43.4 1.8050.0 29.1 20.9 1.1750.0 36.0 14.0 0.8870.0 27.9 2.1 0.16

C14 50.0 36.8 13.2 0.82C16 50.0 43.9 6.1 0.44C18 50.0 37.7 12.3 0.75a

-CHi2 -CH-CH2 -CH-CH2 -CH-

j-

R: A

b VAPI, 4-Vinyl alkylpyridinium iodide

with acetone, and dried under vacuum. Ithis reaction was 250 g (83%).The copolymer (50 g) was added to a p

of an alkyl iodide in ethanol (200 ml). I(moles) of alkyl iodide in the solution wasof the combined pyridines in the copomixture was heated at 100'C under refliInsoluble APyI that formed yellow pearls ied, thoroughly washed with ethanol and tiand dried naturally. The amounts of Acopolymer were determined by argentocompositions of insoluble APyIs and thetions are given in Table 1.

Soluble APyIs were synthesized accormethod of Kourai et al. (10), and the anal)are shown in Table 2.

Preparation of microbial cell suspensiorcoccus aureus 209P, Escherichia coli K-12and Micrococcus luteus ATCC 4698 were uSalmonella typhimurium DB21, Klebsielniae OUT 8017, Pseudomonas aerugin17653, Sarcina lutea, Serratia marcesce?

utilis OUT 6020, Hansenula anomala OUSaccharomyces cerevisiae OUT 7020 wethe antimicrobial spectrum. All of thesemaintained in our laboratories.

Bacteria were cultivated in 10 ml of nutr

APyla 37'C for 18 h, and yeasts were grown in 20 ml of potatodextrose broth at 30'C for 48 h. Gram-positive bacte-

Abbreviation ria, E. coli, and yeasts were aseptically collected on amembrane filter (HAWP 04500; Millipore Corp.) byfiltration and washed with 50 ml of sterilized deionized

C8(50) Other bacteria collected and washed twice

C120(0)with sterilized deionized by centrifugation

C12(30) (3,000 x g, 10 min). Collected cells were suspended in

C12(50)A 9 ml of sterilized deionized water and then diluted to a

C12(50)A prescribed cell concentration with cell suspendingC12(70)B medium. Each suspension was immediately used forC17(50) the antimicrobial tests for insoluble and soluble APyls.C14(50) Antimicrobial tests for insoluble and soluble APyIs.

C16(50) The treatment system for insoluble APyIs is shown inC18(50) Fig. 1. Each insoluble APyI packed in a column was

treated with 60% ethanol to make the agent hydrophil-ic because of the hydrophobicity of the agent. The

Alkyl chain agent was then washed with 1,500 ml of sterilized(C8 '- C I 8) deionized water. A cell suspension was passed

through the column at a constant flow rate and a

prescribed temperature, and the effluent was collectedin an aseptic test tube. Subsequently, the flow rate waschanged to change the residence time, and the sameprocedures were repeated at various flow rates. About

rhe yield of 50 ml of cell suspension was treated at each residencetime, and a total of about 300 ml of cell suspension was

iure solution treated during one series of experiments.['he amount The antimicrobial test for the APyI solutions wasequal to that performed as follows. Five milliliters of the cell sus-lymer. The pension was poured into a 300-mi flask containing 95ix for 12 h. ml of an APyI solution, which had been kept at thewas separat- prescribed temperature. Samples, 10 ml, of the cellhen acetone, suspension were removed at 1, 5, 10, and 15 min afterkPyI in the the start of incubation.)metry. The The sample with continuous treatment was dilutedir abbrevia- with sterilized deionized water. The sample in the

soluble APyI experiment was diluted with 10% steril-rding to the ized polypeptone solution to neutralize the antimicro-ttical results bial activity of the APyI. A 0.1-ml portion of diluted or

undiluted sample was spread on a nutrient agar plate ini. Staphylo- the case of bacteria or on a potato dextrose agar plate2 OUT 8401, in the case of yeasts. The plates were incubated atised mainly. 37'C for 24 to 72 h for bacteria or at 30'C for 48 h for(la pneumo- yeasts, and the antimicrobial activities of insoluble andiosa ATCC soluble APyIs were assessed by the reduction inns, Candida microbial survivors.IT 6079, and In the case of soluble APyIs, the minimum bacteri-re used for cidal concentration was defined as the minimum con-

strains are centration of APyI at which no surviving cells weredetected after 15 min of incubation at 106 cells per ml

ient broth at and 30°C.

TABLE 2. APyI and analysis resultsAnalysis (%)

Alkyl HNM C) Yieldchain C H N mp (C) (

Found Calculated Found Calculated Found Calculated

C8 49.12 48.91 6.98 6.95 4.28 4.39 49-50 67C1O 51.96 51.88 7.66 7.55 3.98 4.03 54-55 57C12 54.30 54.40 7.92 8.06 3.76 3.73 87-88 55C14 56.45 56.57 8.47 8.50 3.56 3.47 94.5-95 46C16 58.48 58.46 8.92 8.88 3.55 3.24 100-100.5 37C18 60.34 60.12 9.37 9.21 3.17 3.05 102-102.5 57

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INSOLUBLE APyI 1043

3

12

0-i 1

Lii 7LIFIG. 1. Schematic diagram of continuous treat-

ment system for cell suspensions. 1, Column; 2, insol-uble agent; 3, microtube pump; 4, circulater; 5, cellsuspension; 6, water bath; 7, magnetic stirrer.

RESULTSSelection of the most active insoluble APyI. The

composition of the insoluble APyI would affectthe antibacterial activity. Therefore, nine kindsof insoluble APyI were synthesized. InsolubleAPyIs, which had various DVB contents and thesame alkyl chain length (C12), showed differentquaternized ratios of VP (Table 1). Then theeffect ofDVB content on the antibacterial activi-ty of the insoluble APyI against S. aureus (106cells per ml) was investigated with 1.0 g each ofC12(10), C12(30), C12(50)A, and C12(70), whose

2

;21I-in0

F0)Inato _1-I

-2

10 30 50 70DV\B content(1.)

FIG. 2. Antibacterial activities of C12(10), C12(30),C12(50)A, and C12(70) (mesh, 80 to 120) against S.

aureus. Cells were suspended in deionized water at 106cells per ml and treated with 1.0 g of each insolubleagent at 30°C. The residence times were as follows: 0,10 s; A, 30 s; O, 60 s; V, 120 s.

8 10 12 14 16 18Carbon number of alkyl chain

FIG. 3. Minimum bactericidal concentration(MBC) of soluble APyI against S. aureus. MBC wasdefined as the minimum concentration at which nosurvivors were detected after 15 min of incubation at300C.

particle sizes were 80 to 120 mesh. C12(50)A hadthe highest activity, and the activity of theothers decreased in the order, C12(30) > C12(10)> C12(70) (Fig. 2).

It has been known that the antibacterial activi-ty of an APyI depends on its alkyl chain length(10). Also, in this experiment, soluble APyI witha longer alkyl chain showed a lower minimumbactericidal concentration against S. aureus(Fig. 3). In the case of insoluble APyIs, such aneffect would also be observed. A 1.0-g amount ofan insoluble APyI, with a particle size rangingfrom 32 to 80 mesh and a DVB content of 50%,was used to investigate the effect of alkyl chainlength on antibacterial activity against S. aureus(10c cells per ml) at 30°C. C8(50), C10(50), andC12(50)A had high antibacterial activities, butC14(50), C16(50) and C18(50) showed no activity(Fig. 4).From the above results, C12(50) was selected

for the following investigations.Durability of antibacterial activity of C12(50).

In the above-described experiments, only about300 ml of cell suspension was passed through thecolumn packed with an insoluble APyI duringone experiment. The cell suspension of S. aure-us (106 cells per ml), therefore, was continuouslytreated with 1.0 g of C12(50)A (80 to 120 mesh;bed volume, 2.4 cm3) with a residence time of 3min at 30°C. The antibacterial activity ofC12(50)A was evaluated when a new cell suspen-sion was substituted for the old one every 24 h.The activity of C12(50)A decreased remarkablyafter 24 h (Fig. 5). After 96 h, C12(50)A waswashed with deionized water, but no recovery of

:1'

I.

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Carbon number of alkyl chainFIG. 4. Effect of alkyl chain length on the antibac-

terial activity of 1.0 g of C,(50) (mesh, 32 to 80) againstS. aureus. The treatment conditions were the same as

in the legend to Fig. 2. The residence times were as

follows: 0, 5 s; A, 30 s; L, 120 s.

activity was observed. C12(50)A completely re-covered the antibacterial activity upon ethanoltreatment. When refreshed C12(50)A was usedagain for the continuous treatment of a cellsuspension, the activity decreased in the samemanner as for fresh C12(50)A (Fig. 5). Further-more, deionized water instead of the cell suspen-

0 40 80 0 20 0 20

Treatment time (hr)

FIG. 5. Decrease and recovery of the antibacterial

activity of C12(50)A (mesh, 80 to 120) against S.

aureus. Cells were suspended in deionized water at 106

cells per ml and treated with 1.0 g of C12(50)A with a

residence time of 3 min at 30°C. a, C12(50) was washed

with 1,500 ml of deionized water; b, C12(50) was

washed with 1,500 ml of ethanol and subsequently

with 1,500 ml of deionized water.

sion was passed continuously through the col-umn, and the cell suspension was treated onlywhen the activity of C12(50)A was evaluated. Asa result, the decrease in activity with only deion-ized water was observed to be to the samedegree as shown in Fig. 5.

C12(50)A was refreshed with ethanol aftertreatment of about 300 ml of cell suspension, andit was used again for the antibacterial test. Theseprocesses were repeated several times. The ac-tivity of C12(50)A was the same on the thirdrepeated use, but decreased on the fourth. Onrepeated use after the fifth time, no activity wasobserved (Fig. 6). C12(50)A, which did not re-cover activity after ethanol treatment, recoveredcomplete antibacterial activity after 1.0 NNaOH treatment.

In the following experiments, fresh C12(50)Aor -B was used only once without refreshmentwith ethanol or 1.0 N NaOH.

Variance of antibacterial efficiency based onamount of C12(50) and its particle size. Whetheror not the antibacterial efficiency of C12(50)depends on the amount of the agent was investi-gated. The amount of C12(50)A was increased inincrements of 0.25 g, from 1.0 to 2.0 g inclusive.Its particle size was always 60 to 80 mesh. Theantibacterial efficiency of C12(50)A against S.aureus (106 cells per ml) at 30°C became higherwith increasing amount used (Fig. 7). This wasespecially significant at more than 1.5 g ofC12(50)A.

2

0

0-J -2

-3

0 1 2 3Residence time(min)

FIG. 6. Change in antibacterial activity of C12(50)A(mesh, 80 to 120) against S. aureus due to number oftreatments. A 1.0-g portion of C12(50)A was washedwith 1,500 ml of ethanol and then with 1,500 ml ofdeionized water before each measurement of the activ-ity. The treatment conditions were the same as in thelegend to Fig. 2. Symbols: 0, lst; *, 2nd; A, 3rd; A,4th; 0, Sth; *, 6th treatment.

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u,-2

-3

1.0 1.5 2.0Amount of C12(50) (g)

FIG. 7. Effect of the amount of C12(50)A (mesh, 60to 80) on antibacterial efficiency against S. aureus. Thetreatment conditions were the same as in the legend toFig. 2. The residence times were as follows: 0, 10 s;A, 30 s; O, 60 s; V, 120 s.

The particle size of C12(50)A would affect theantibacterial efficiency because of the contactarea of bacteria with the solid agent. The smallerthe particle size was, the higher the antibacterialefficiency against S. aureus (106 cells per ml) at30°C (Fig. 8). In the case of C12(50)A, whoseparticle size was 120 to 150 mesh, no survivingcells were detected.

Antimicrobial spectra. Antimicrobial spectraof C12(50)A and laurylpyridinium iodide (LPyI)solution were examined at 30°C. In this experi-ment, the cell concentration was approximately106 cells per ml, and 1.5 g of C12(50)A (80 to 120mesh) and 100 ,uM LPyI aqueous solution wereused (Table 3). C12(50)A showed high antimicro-bial activity against gram-positive bacteria andalso showed some activity against gram-negativebacteria and yeasts. C12(50)A had higher activityagainst S. aureus, P. aeruginosa, Serratiamarcescens, H. anomala, and Saccharomycescerevisiae than did the LPyI solution on thebasis of the activities against E. coli, but itshowed lower activity against Salmonella typhi-murium and C. utilis. The activities against otherorganisms were nearly equal for C12(50)A andthe LPyI solution. Therefore, E. coli and M.luteus were used in the following experimentsbecause the antimicrobial activities of C12(50)Aand LPyI solution against these bacteria were

almost the same.Effect of temperature on antibacterial efficien-

cy. The effect of temperature on the antibacterialefficiencies of C12(50)B and LPyI solution was

examined. The cell concentrations of the bacte-ria tested were 106 cells per ml; 1.1 g of C12(50)B(32 to 80 mesh) and 55 ,uM LPyI solution were

used against E. coli, and 0.5 g of C12(50)B (32 to

80 mesh) and 10 ,uM LPyI solution were usedagainst M. luteus. In the following experiments,the same amounts and concentrations of theagents were used. These were determined as theamounts and concentrations at which changes inthe antibacterial efficiencies under various con-ditions were recognized.The results for E. coli and M. luteus are

shown in Fig. 9 and 10, respectively. Bothagents showed higher antibacterial activities at ahigher temperature, and surviving cells of E. coliwere not detected at 35°C even with a 10-sresidence time in the case of C12(50)B. Theantibacterial efficiency of the LPyI solutionshowed the same dependence on temperatureagainst E. coli and M. luteus, but the sensitivityof E. coli to C12(50)B was more affected bytemperature than was that of M. luteus.

Effect of cell concentration on survivors. Fig-ures 11 and 12 show the effect of cell concentra-tion on the antibacterial activities of C12(50)Band LPyI solution against, respectively, E. coliand M. luteus at 30°C. C12(50)B had higherantibacterial activity at a lower cell concentra-tion, and no survivors were detected at a cellconcentration of 105 per ml. The LPyI solutionshowed the same effect as C12(50)B against M.luteus, but the influence of cell concentrationwas not observed against E. coli. From theseresults, it was concluded that the antibacterialactivities of C12(50)B and LPyI solution wereinfluenced more easily by the cell concentrationof M. luteus than by that of E. coli.The influence of cell concentration seemed to

be related to the contact probability betweencells and C12(50). Therefore, a mixed suspensionof dead and living cells was used to test forantibacterial activity of C12(50)B. A cell suspen-

-2

Residence time (min)FIG. 8. Effect of particle size on antibacterial effi-

ciency of C12(50)A against S. aureus. The treatmentconditions were the same as in the legend to Fig. 2.Symbols: 0, 32 to 60; A, 60 to 80; O, 80 to 120 mesh.

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TABLE 3. Antimicrobial spectra of C12(50)A and LPyI solution"

C12(50) at residence time (s): LPyI at incubationMicrobe ( time (min):

10 30 60 120 180 5 15

Staphylococcus aureus +b - - - - ++ ++Micrococcus luteus - - - - - - -Sarcina luteaEscherichia coli ++ ++ + + - + +Salmonella typhimurium ++ + + + +Klebsiella pneumoniae ++ ++ ++ ++ + ++ ++Pseudomonas aeruginosa + - - - - + + ++Serratia marcescens + + + + - - ++ ++Candidautilis +++ +++ +++ ++ ++ ++Hansenula anomala +++ ++ + + - + + + +++Saccharomyces cerevisiae +++ ++ + - - + + + +++

a Each microbe was suspended in deionized water at approximately 106 cells per ml and treated with 1.5 g ofC12(50)A (mesh, 80 to 120) and 100 ,uM LPyI solution at 30°C.

b + + +, 0 < log survivors (percent) _ 2; + +, -2 < log survivors (percent) ' 0; +, log survivors (percent) '-2; -, no survivors.

sion of M. luteus was autoclaved at 121°C for 5min and then separated into dead cells andfiltrate with a membrane filter (HAWP 04500;Millipore Corp.). Dead cells were added to aliving cell suspension of M. luteus. The finalconcentrations of living and dead cells were 106and 107 per ml, respectively. The suspensionwas treated with C12(50)B (Fig. 13). The antibac-terial activity of C12(50)B decreased remarkablycompared with the case of no addition at 106living cells per ml. The dead cell suspensionmixed with the filtrate also decreased the anti-bacterial efficiency of C12(50)B, and the numberof survivors was almost the same as in the caseof the suspension of 107 living cells per ml (Fig.13). These results showed that the antibacterial

activity of C12(50)B was influenced by the cellconcentration, whether the cells were living ordead, and by materials leaking from the cells.Experiments with E. coli and LPyI solutionwere not performed.

Effect of cell suspending medium on survivors.The antibacterial activity of C12(50)B was de-creased by the materials leaking from dead cells(Fig. 13). Therefore, the effect of cell suspendingmedium was examined at 30°C and 106 cells perml. NaCl (0.9%) solution, 0.1% glucose solution,0.1% bovine albumin solution, and deionizedwater were used as cell suspending media. Theresults are shown in Fig. 14 and 15. The antibac-terial activities of C12(50)B and liquid LPyI werehighest in the case of deionized water and de-

0 1 2 0 5 10Residence time(min) Incubation time(min)

FIG. 9. Effect of temperature on antibacterial activities of Cl2(50)B (mesh, 32 to 80) (A) and LPyI solution (B)against E. coli. Cells were suspended in deionized water at 106 cells per ml and treated with 1.1 g of C12(50)B and55 ,uM LPyI solution at each temperature. Symbols: A, 20°C; O, 25°C; 0, 30°C; V, 35°C.

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0

-2

0 30 60 0 5 10Residence time(sec) Incubation time(min)

FIG. 10. Effect of temperature on antibacterial activities of C12(50)B (mesh, 32 to 80) (A) and LPyI solution(B) against M. luteus. Cells were suspended in deionized water at 106 cells per ml and treated with 0.5 g ofC12(50)B and 10 FM LPyI solution at each temperature. Symbols are the same as in Fig. 9.

creased in the following order: 0.1% glucose >0.1% bovine albumin > 0.9% NaCl.When a 60-,ug/ml bovine albumin solution was

passed through a column packed with 1.1 g ofC12(50)B, the concentration of bovine albuminin the effluent decreased to 45 ,ug/ml. It might beconcluded that the decrease in activity resultedfrom the adhesion of bovine albumin toC12(50)B.Although the effect of pH on the antibacterial

activity of C12(50)B was examined with 0.01 Msodium-potassium phosphate buffer at pH 6.0,

7.0, and 8.0, the activity was not influenced bypH. However, the activity decreased remark-ably in each phosphate buffer (data not shown).

DISCUSSIONIn these experiments, released APyI from

insoluble agents could not be detected in treatedcell suspensions by the orange II-chloroformmethod (1). With this method it was possible todetect 0.1 ,ug of APyI per ml. Therefore, theinsoluble APyIs, differing from quaternary am-

77L

2

O 1 2 0 5 10Residence time(min) Incubation time (min)

FIG. 11. Effect of cell concentration on antibacterial activities of C12(50)B (mesh, 32 to 80) (A) and LPyIsolution (B) against E. coli at 300C. Cells were suspended in deionized water at various concentrations. Theamount of C12(50)B and the concentration of the LPyI solution were the same as in the legend to Fig. 9. Initialcell concentrations were as follows: O, 3 x 108; O, 108; V, 3 x 107; A, 107; 0, 106; 0 105 cells per ml.

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E 70

L-

U)

o 5

5 4In

23

(A) (B)

1:3- {, r

'0 30 60 0 5 10Residence time (sec) Incubation time (min)

FIG. 12. Effect of cell concentration on antibacterial activities of C12(50)B (mesh, 32 to 80) (A) and LPyIsolution (B) against M. luteus at 300C. Cells were suspended in deionized water at various cell concentrations.The amount of C12(50)B and the concentration of the LPyl solution were the same as in the legend to Fig. 10.Initial cell concentrations were as follows: O, 108; A, 107; V, 5 x 10'; 0, 10' cells per ml.

monium salts insolubilized by the ionic bondmethod (12, 13), were successful in that nosoluble agent was detected. Because the numberof survivors in the cell suspension did not de-crease in the case of treatment with the VP-DVBcopolymer, it was considered that bacterial cells

2

.o o\

0

-2

0 30 60Residence time (sec)

FIG. 13. Effect of dead cells and materials leakingfrom dead cells on antibacterial activity of C12(50)B(mesh, 32 to 80) against M. luteus. Living cells (106cells per ml) were suspended in solutions containingdead cells or the leaked materials and were treatedwith 0.5 g of C12(50)B at 300C. Symbols-solutionscontaining: 0, 107 dead cells per 1 ml of living cellsuspension; A, the materials that had leaked from 107dead cells per 1 ml of living cell suspension; O, 107dead cells and the leaked materials per 1 ml of livingcell suspension; 0, control (106 cells per ml), noaddition; A, control (107 cells per ml), no addition.

were not filtered by these insoluble agents dur-ing continuous treatment.Although C12(10) and C12(30) had higher LPyI

contents than did C12(50) (Table 1), C12(10) andC12(30) showed lower antibacterial activities.LPyI may exist not only on the surface but alsoon the inner part of the particles because thesepolymers have the three-dimensional structureof a network. Only LPyI on the surface, howev-er, may be able to come into contact withbacteria because the size of cells is remarkablylarger than that of the network. C12(10) andC12(30) have less DVB than C12(50), so thepenetration of lauryl iodide into the inner part ofthe former may be easier than for the latter, andthe lauryl iodide binding sites on the inner partof the particles will significantly increase withdecreasing DVB content. Therefore, the effec-tive LPyI in C12(50) will be much more than thatin C12(10) and C12(30), which have the plentifultotal LPyI. This may be the reason why C12(50)showed higher antibacterial activity than C12(10)and C12(30). C12(70) had the most DVB, but theamount of LPyI in it was too low to exhibit theantibacterial activity.

Kourai et al. (10) reported that, in an aqueousAPyI solution, the antibacterial activity in-creased with increasing alkyl chain length. Thiswas also observed in our experiment (Fig. 3),but in our experiment, for insoluble APyIs, theantibacterial activities of C8(50), C10(50), andC12(50) were higher than those of C14(50),C16(50), and C18(50). It was concluded that thiswas due to the hydrophobic extent of theseinsoluble APyIs and the APyI content of theseagents (Table 1). Hydrophobicity of these insol-

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O 1 2 0 5 10Residence time(min) Incubation time (min)

FIG. 14. Effect of cell suspending medium on antibacterial activities of C12(50)B (mesh, 32 to 80) (A) andLPyI solution (B) against E. coli at 30°C. Cells were suspended in various solutions at 106 cells per ml. Theamount of C12(50)B and the concentration of the LPyI solution were the same as in the legend to Fig. 9. Symbols:0, deionized water; A\, 0.1% glucose; O, 0.1% bovine albumin; V, 0.9% NaCl.

uble agents was assessed by the ethanol concen-tration needed for their hydrophilization, andthe insoluble agents which had longer alkylchains needed higher ethanol concentrations.When C12(50) was continuously used for a

long period, the antibacterial activity decreased,but the activity was recovered by washing withethanol. Also, when deionized water was usedinstead of cell suspension, the activity decreasedto the same level as with cell suspension. Fromthis, it was concluded that this decrease ofactivity was caused by adhesion of dissolved airto C12(50) because of the hydrophobicity of

C12(50) and that the adhesion of air inhibited thecontact of cells with C12(50). When C12(50) wasused repeatedly, the activity could not be recov-ered by washing with ethanol, but it could berecovered by washing with 1.0 N NaOH. There-fore, it was suggested that this decrease inactivity was caused by adhesion of cells andsome materials which could be released fromC12(50) by NaOH treatment to C12(50).From the results in Fig. 7 and 8, the antibac-

terial activity of the insoluble agents was seen todepend on the total surface area of the insolubleagents.

Residence time(sec) Incubation time (min)FIG. 15. Effect of cell suspending medium on antibacterial activities of C12(50)B (mesh, 32 to 80) (A) and

LPyI solution (B) against M. luteus. The amount of C12(50)B and the concentration of the LPyI solution were thesame as in the legend to Fig. 10. Other conditions and symbols were the same as in the legend to Fig. 14.

VOL. 43, 1982

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1050 NAKAGAWA ET AL.

It is usually recognized that gram-positivebacteria have higher sensitivities to quaternaryammonium salt solutions than gram-negativebacteria and molds. However, S. aureus showedlow sensitivity and Salmonella typhimuriumshowed high sensitivity (Table 3). S. aureusshowed a much higher minimum bactericidalconcentration to APyI (C8, C1o, and C12) solu-tions than to APyI (C14, C16, and C18) solutions(Fig. 3). It was therefore suggested that this lowsensitivity of S. aureus to the LPyI solution wasspecific. C12(50) was an effective agent for gram-positive bacteria, and P. aeruginosa, Serratiamarcescens, H. anomala, and Saccharomycescerevisiae showed higher sensitivity to C12(50)than to liquid LPyI (Table 3). The latter fourmicrobes had higher resistance to aqueous LPyIthan did E. coli. From these results it wassuggested that the mode of action of C12(50) wasnot the same as that of liquid LPyI.The cell concentration always influenced the

antibacterial efficiencies of C12(50) and LPyIsolution except for the combination of LPyIsolution and E. coli. The efficiency decreasedwith increasing cell concentration regardless ofwhether the cells were living or dead. Thisphenomenon indicated that every cell interferedwith the contact of other cells with C12(50).Therefore, the problems of cell concentrationmeant the probability of contact of each cell withC12(50).Glucose and bovine albumin reduced the anti-

bacterial efficiencies of C12(50) and LPyI solu-tion. The same phenomenon was observed forthe supernatant from an autoclaved cell suspen-sion. This reduction might be due to the shield-ing of the active site of C12(50) by these organicsubstances because of the adsorption of bovinealbumin by C12(50). Phosphate buffer and NaClalso decreased the antibacterial efficiencies ofC12(50) and LPyI solution. These are dissociat-ing compounds, so the dissociated ions mightprevent the association of cells with the agents.These results showed that some insoluble

agents had high antimicrobial activity, but theseagents were poor in durability of the activity.For general application, it is necessary to inves-tigate methods of preserving the activity. Fur-thermore, the antimicrobial characteristics ofC12(50) and LPyI solution were almost the same,but a few differences were found. Such differ-ences might result from the difference in antimi-crobial mechanisms of the agents. Investigationof the mechanism of action is also important forapplication. These problems are being investi-gated in our laboratories.

LITERATURE CITED

1. Few, A. V., and R. H. Ottewill. 1956. A spectrophotomet-ric method for the determination of cationic detergents. J.Colloid Sci. 11:34-38.

2. Gilmour, M. N., and E. K. Wicksell. 1972. Bactericidaland sporicidal activity of a quaternary ammonium resin-triiodide complex. Antimicrob. Agents Chemother. 2:417-418.

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

4. Isquith, A. J., and C. J. McCollum. 1978. Surface kinetictest method for determining rate of kill by an antimicrobialsolid. Appl. Environ. Microbiol. 36:700-704.

5. Jono, K., T. Yamano, T. Fujimoto, and Y. Eguchi. 1977.Bactericidal action of active carbon coated with silver andits application to water purifiers. J. Takeda Res. Lab.33:9-18.

6. Kao, I. C., L. E. Erickson, W. E. Craig, and L. T. Fan.1973. Effects of temperature and resin particle on theperformance of a triiodide disinfection column. J. Fer-ment. Technol. 51:159-161.

7. Kao, I. C., D. E. Robker, L. T. Fan, A. Snyder, and L. E.Erickson. 1972. Analysis and properties of a quaternaryammonium triiodide ion exchange sterilization process. J.Ferment. Technol. 50:438-445.

8. Kennedy, J. F., and H. Cho Tun. 1973. Active insolubi-lized antibiotics based on cellulose and cellulose carbon-ate. Antimicrob. Agents Chemother. 3:575-579.

9. Kennedy, J. F., J. Epton, and G. R. Kennedy. 1973.Poly(N-acryloyl-4-and -5-aminosalicylic acids). II. Anti-bacterial properties and uses for the preparation of active,insoluble antibiotics. Antimicrob. Agents Chemother.3:29-32.

10. Kourai, H., T. Horie, K. Takeichi, and I. Shibasaki. 1980.The antimicrobial characteristics of quaternary ammoni-um salts and their alkyl chain length. J. Antibact. Anti-fung. Agents 8:191-199.

11. LaPorte, D. C., K. S. Rosenthal, and D. R. Storm. 1977.Inhibition of Escherichia coli growth and respiration bypolymyxin B covalently attached to agarose beads. Bio-chemistry 16:1642-1648.

12. Nakagawa, Y., Y. Inoue, T. Tawaratani, and I. Shibasaki.1979. Bactericidal activity of insoluble cetylpyridiniumchloride-amberlite IRC50-complex under continuous sys-tem. J. Antibact. Antifung. Agents 7:T551-T556.

13. Nakagawa, Y., T. Tawaratani, and I. Shibasaki. 1979.Microbicidal activity of insoluble quaternary ammoniumsalt-cation-exchange resin complex. J. Antibact. Anti-fung. Agents 7:T511-T518.

14. Suzuki, T., and L. T. Fan. 1979. A disinfection mecha-nism of triiodide type anion-exchanger. J. Ferment. Tech-nol. 57:578-581.

15. Tawaratani, T., H. Tsujii, and I. Shibasaki. 1976. Studieson the sterilization of bacteria by iodine-quaternary am-monium resin complex. J. Ferment. Technol. 54:158-165.

16. Taylor, G. F. 1970. Survival of Gram-negative bacteria onplastic compounded with hexachlorophene. Appl. Micro-biol. 19:131-133.

17. Taylor, S. L., L. R. Fina, and J. F. Lambert. 1970. Newwater disinfectant: an insoluble quaternary ammoniumresin-triiodide combination that releases bactericide ondemand. Appl. Microbiol. 20:720-722.

18. Walters, P. A., E. A. Abbott, and A. J. Isquith. 1973.Algicidal activity of a surface-bonded organosilicon qua-ternary ammonium chloride. Appl. Microbiol. 25:253-256.

APPL. ENVIRON. MICROBIOL.

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