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Antibacterial Properties of TannicAcid and Related Compoundsagainst the Fish PathogenCytophaga columnarisGuojing Zhao a , King-Thom Chung a , Kimberly Milow a ,Wenxian Wang a & S. Edward Stevens Jr. aa Department of Microbiology and Molecular CellSciences , The University of Memphis , Memphis,Tennessee, 38152, USAPublished online: 09 Jan 2011.

To cite this article: Guojing Zhao , King-Thom Chung , Kimberly Milow , Wenxian Wang & S.Edward Stevens Jr. (1997) Antibacterial Properties of Tannic Acid and Related Compoundsagainst the Fish Pathogen Cytophaga columnaris , Journal of Aquatic Animal Health, 9:4,309-313, DOI: 10.1577/1548-8667(1997)009<0309:APOTAA>2.3.CO;2

To link to this article: http://dx.doi.org/10.1577/1548-8667(1997)009<0309:APOTAA>2.3.CO;2

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Journal of Aquatic Animal Health 9:309-313, 1997© Copyright by the American Fisheries Society 1997

Antibacterial Properties of Tannic Acid andRelated Compounds against the Fish Pathogen

Cytophaga columnaris

GUOJING ZHAO, KING-THOM CHUNG, KIMBERLY MILOW, WENXIAN WANG,AND S. EDWARD STEVENS, JR.1

Department of Microbiology and Molecular Cell SciencesThe University of Memphis, Memphis, Tennessee 38152, USA

Abstract.-Tannic acid, gallic acid, and propyl gallateexhibited inhibitory activity as demonstrated by the agardilution assay against Cytophaga columnaris (=Bacilluscolumnaris, Chondrococcus columnaris, Flexibacter co-lumnaris, or Flavobacterium columnare), a ubiquitousgliding fish pathogen, at 150, 275, and 300 ug/mL, re-spectively, in modified Shieh medium, at a low-bacte-rial-inoclum density of 103-4 colony-forming units/mL .Methyl gallate was not effective at the highest concen-tration tested (500 ug/mL) . The minimum inhibitoryconcentrations (MICs) of gallic acid, methyl gallate, andpropyl gallate were lower in natural pond water than inmodified Shieh medium, whereas the MIC of tannic acidwas the same in both . Tannic acid, a polymeric com-pound with multiple hydroxyl groups, had a greater ca-pacity for binding protein and glycogen by at least ninetimes that of the other test compounds . The results sug-gest that the hydroxyl group availability of tannins isessential for antibacterial activity .

Cytophaga columnaris (=Bacillus columnaris,Chondrococcus columnaris, Flexibacter columna-ris, or most recently, Flavobacterium columnare;Bernardet and Grimont 1989; Staley et al . 1989 ;Bernardet et al . 1996) is a ubiquitous, gliding bac-terium in the aquatic environment. Under normalconditions, C. columnaris is a saprophyte in ponds,but under stressful conditions such as a suddentemperature change, decrease in oxygen supply,dense fish population, contamination by other bac-teria, eutrophic environment, or injuries of the fishbody, the bacterium becomes a serious pathogen(Bullock 1972) that is regarded as responsible forcolumnaris disease in fish (Davis 1923). Colum-naris disease is one of the most common bacterialdiseases worldwide in all freshwater fish species,particularly salmonids and channel catfish (Tuckerand Robinson 1990) . The disease has resulted ingreat economic loss to the fish farming industryin the United States . Control of the spontaneousoccurrence of columnaris epizootics is necessaryfor the healthy growth of the catfish industry . A

possible means for such control may be providedby natural tannins.

Tannins are generally classified into hydrolys-able or condensed tannins . Hydrolysable tannins,commonly referred to as tannic acid (gallotannicacid, gallotannin, or penta-m-digalloyl-glucose),are esters of phenolic acids and a polyol that isusually glucose (Haslam 1989; Scalbert 1991). Be-cause these natural substances are widespread andreadily available in the environment (White 1957)and as food additives, are categorized as "gener-ally recognized as safe" (GRAS) according to theCode of Federal Regulations (USOFR 1989), theantimicrobial activities and chemotherapeutic po-tentials of these compounds have been the subjectof increasing study. Our previous work has shownthat tannic acid and related compounds were in-hibitory to the growth of many microorganismsincluding aquatic bacteria (Chung et al . 1993,1995b), the bloom-causing cyanobacterium Ag-menellum quadruplicatum strain PR-6, and the off-flavor-producing cyanobacterium Nostoc sp . strainMAC (Chung et al . 1995a) .

This paper reports the effect of tannic acid andits derivatives on the growth of the fish pathogenC. columnaris . We also measured the protein pre-cipitation and polysaccharide binding capacities,lipophilicity, and other physicochemical propertiesof these compounds to understand possible mech-anisms for their antibacterial action .

MethodsBacterial strain and culture condition.-The test

organism Cytophaga columnaris was obtainedfrom the National Biological Survey (now the U.S .Geological Survey, Biological Research Service),Marion, Alabama. This representative strain wasspecifically isolated from channel catfish Ictaluruspunctatus and maintained on modified Shieh me-dium at 4°C. In preparation for experiments, C.columnaris was transferred to 2 mL of modifiedShieh broth medium and grown overnight at 32°Cto a concentration of 2 X 107 colony-forming units1 To whom correspondence should be addressed.

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(CFU)/mL through viable plate counting with anabsorbance of 0.2 at 595 nm . Modified Shieh me-dium, pH 7 .4, supported the growth of C. colum-naris and was used for routine cultivation . Themedium (per 100 mL) contained peptone, 500 mg;sodium-acetate, 1 mg ; yeast extract, 50 mg ; BaCl2H2O, 1 mg ; K2HPO4, 10 mg ; KH2PO4, 5 mg ;MgSO4 7H20, 30 mg ; CaCl2 2H20, 0.67 mg ;FeSO4 7H20, 0.1 mg ; and NaHCO3, 5 mg (Shieh

1980 ; Song et al . 1988) .All chemicals, including the test compounds

gallic acid, methyl gallate, propyl gallate, and tan-nic acid, were purchased from Sigma ChemicalCo . (SL Louis, Missouri) unless otherwise speci-fied . The tannin compounds were freshly preparedat specified concentrations by dissolving them indeionized water, adjusting their pH to 7.4 with 1N NaOH, and then filter-sterilizing the solutions .These stock solutions were then used for suscep-tibility tests or for biological activity tests .Agar dilution in Shieh medium.-The agar dilution

method was employed to assay the antibacterial pro-pertites of tannic acid and related compounds (Sand-ven et al . 1993) . Serial two-fold dilutions of gallicacid, methyl gallate, propyl gallate, or tannic acidwere prepared for concentrations ranging from 0.5to 5 .0 mg/mL. A portion (1 .5 mL) of each dilutionwas mixed with 13 .5 mL of modified Shieh agarmedium to give final chemical concentrations of 0.05to 0.5 mg/mL in the agar plates . One milliliter of anovernight culture of the C. columnaris isolate (2 X107 CFU/mL) in modified Shieh medium was seriallydiluted in sterile Butterfield phosphate buffer, pH 7.2(FDA 1992), to approximately 2 X 104 CFU/mL ofbacterial suspension . 50-uL quantity of each of theovernight culture (2 X 107 CFU/mL) and the dilutedculture (2 X 104 CFU/mL) was inoculated onto sep-arately prepared plates to obtain final concentrationsof either 106 CFU/mL (high-inoculum density) or103 CFU/mL (low-inoculum density) . A plate with-out the test compounds was used as a control . Theinoculated plates were incubated at 32°C for 48-72h. The minimum inhibitory concentration (MIC) oftest compounds was defined as the point ofno growthin a serial two-fold dilution sequence .Agar dilution in pond water.-The same pro-

cedure as described above was performed withpond water agar instead of modified Shieh mediumagar. Fresh pond water was obtained from severalaquaculture ponds at different times of the day andthe year (spring and summer) at Shelby Farm inMemphis, Tennessee, about 16 km from our lab-oratory, and agar was then made according to themethods of Chung et al . (1995b) . Pond water agar

without treatment with tannin compounds servedas a control culture, which can support the growthof C. columnaris .

Protein precipitation assay.-The Rickard(1986) method was applied to measure the proteinprecipitation capacities of tannins. A standard pro-tein solution (1 .0 mg/mL) of bovine serum albu-min (fraction 5) was freshly prepared in distilledwater, and a test compound solution (1 .0 mg/mL)was prepared in either distilled water or pond wa-ter . Four milliliters of test solution were added to16 mL of the standard protein solution . The mixedsolutions were agitated on a flask shaker at roomtemperature for 30 min and then centrifuged at8,000 X gravity for 15 min. The protein contentof the supernatants was measured by using the Bio-Rad assay (Bio-Rad Laboratories, Hercules, Cal-ifornia) . The results are expressed as milligramsof protein precipitated by 1 mg of test compound .

Lipophilicity assay. -Thin-layer chromatogra-phy (Smith and Feinberg 1965; Cain et al . 1974)was used to test the lipophilicity of tannin com-pounds . Silica gel plates (Merck Si02 F254 ; What-man International Ltd., Maidstone, UK) were usedwith N-BuOH-0.1 N NH4Cl (1 :1, volume : vol-ume) as the solvent system . Samples (30 uL) wereapplied and allowed to migrate until the solventfront had moved approximately 10 cm . The mi-gration of spots was measured, and the lipophil-icity (Rf) values were then calculated (Smith andFeinberg 1965). The lower the Rf value, the morelipophilic the compound .

Polysaccharide binding assay.-Nelson's test(Wharton and McGorty 1982) was performed toassess the concentration of reducing sugar afteracid hydrolysis of glycogen (bovine liver type 9) .Briefly, after tannin compounds were mixed withglycogen, 2 mL of the supernatant was vortexedwith 2 mL of 4 M HCI . An aliquot of 0.5 mL ofthe mixture was transferred to a test tube contain-ing 2.5 mL 1 M K2HPO4, and the volume wasbrought to 10 mL with distilled water. One mil-liliter of the hydrolysate was mixed with 1 mL ofNelson's reagent C, a mixture of 25 mL of reagentA (containing 12 .5 g of Na2CO3, 12 .5 g of NaKtartrate, 10 g of NaHCO3, and 10 g of Na2SO4 in500 mL of distilled water), and 1 mL of reagentB (containing 15 g of CuSO4.5H2O and 1 drop ofH2SO4 in 100 mL of distilled water) . The mixturewas placed in a boiling water bath for 20 min.After the mixture cooled, 1 mL of arsenomolyb-date reagent (containing 25 g of ammonium mo-lybdate, 21 mL of concentrated H2SO4, and 3 gof Na2HASO4 7H2O in 475 mL of distilled water)

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was added to the tubes, which were allowed tostand for 2 min before the volume was brought to10 mL with distilled water. The absorbance of thesamples was measured at 510nm with a Spectronic20 colorimeter. The amount of reducing equiva-lents was calculated from the D-glucose standardcurve (Wharton and McGorty 1982). Results wereexpressed as milligrams of glycogen precipitatedby 1 mg of test compound . All the assays wererepeated two or three times and the results wereaveraged .

ResultsWith the agar dilution assay method, gallic acid,

propyl gallate, and tannic acid were found to in-hibit the growth of C. columnaris on both modifiedShieh medium and pond water agar plates (Table1) . At low-inoculum density (103-4 CFU/mL), the

MICs of gallic acid, propyl gallate, and tannic acidwere 275, 300, and 150 ug/mL, respectively, onmodified Shieh medium agar. Methyl gallate wasnot effective up to at the highest concentrationtested (500 ug/mL) . At high-inoculum density(106-7 CFU/mL), the MICs for gallic acid and tan-nic acid were 425 and 225 ug/mL, respectively .Neither methyl gallate or propyl gallate was ef-fective at concentrations up to 500 ug/mL.

In pond water agar, the antibacterial activity ofgallic acid, methyl gallate, and propyl gallate in-creased dramatically, but that of tannic acid re-mained the same as in modified Shieh medium . Atlow-inoculum density, the inhibitory activityranged from 75 to 200 ug/mL, whereas at high-inoculum density, it ranged from 200 to 350 ug/mL. Compared with the inhibitory effects exertedin modified Shieh medium, the anticytophagal ac-tivity of gallic acid, methyl gallate, and propylgallate increased by at least 50%. Tannic acid,however, did not show any demonstrable increasein its inhibition of C. columnaris in pond wateragar (Table 1) . Our experimental results were con-sistent in pond water tests with different pond wa-ter samples.The results of the lipophilicity assay as deter-

mined by Rf values (Table 2) indicated that gallicacid and tannic acid were more lipophilic thanmethyl gallate and propyl gallate . Tannic acid wassignificantly stronger in protein-binding capacitythan gallic acid, methyl gallate, or propyl gallate .One milligram of tannic acid precipitated 3 .184mg of protein (approximately 10 times the proteinprecipitation of methyl gallate, 11 times that ofpropyl gallate, and 17 times that of gallic acid) .Tannic acid also had a significantly greater capac-

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312

ZHAO ET AL .

ity for binding glycogen (approximately 9 timesthe glycogen-binding capacity of gallic acid and41 times that of methyl gallate) . Propyl gallate hadno detectable glycogen-binding capacity . TheMICof tannic acid was 40-240 ug/mL against eightother aquatic bacteria and C. columnaris (Chunget al . 1995a, 1995b) .When biological activities of test compounds

were measured in natural pond water, significantlydifferent values for protein- and glycogen-bindingcapacities were obtained (Table 3) . Natural pondwater did not affect the lipophilicity (Rf) of thesecompounds. However, the protein-binding capac-ity of gallic acid and methyl gallate increased inpond water, that of tannic acid decreased dramat-ically, and that of propyl gallate decreased slightly .The glycogen-binding capacity of methyl gallateand propyl gallate increased, but that of tannic aciddecreased in natural pond water. Glycogen was notbound by gallic acid .

DiscussionThe 3,4,5-trihydroxy benzene ring (Figure 1) is

the core structural moiety of all hydrolysable veg-etable tannins. The addition of a 1-carboxyl grouponto this core structure yields a typical monomerichydrolysable tannin . Esterification with otherchemical groups such as alkyls or sugars providesthe diversity and complexity of tannin compounds(Table 2) .Our results suggest that the protein precipitation

ability and polysaccharide-binding capacity of tan-nin compounds are attributable to the availabilityof the hydroxyl groups on the core structure of thetannin molecules . These hydroxyl groups may pro-duce hydrogen bonds. Experimental evidenceshown in Table 2 supports the notion that tannicacid, a polymer, was more effective than mono-mers such as gallic acid, methyl gallate, and propylgallate in forming complexes with macromole-cules in biological systems (Field et al . 1989).Such binding may interfere with many enzymaticfunctions in bacteria and with the surface patterns

of cells, which may be crucial for control of cel-lular growth (Pate and Ordal 1967).

This is also supported by the fact that the anti-C. columnaris activity of tannic acid was not foundto have any demonstrable increase in pond wateras contrasted with gallic acid, methyl gallate andpropyl gallate . The pond water strongly reducedthe ability of tannic acid to bind protein and gly-cogen (Table 3), resulting in the partial or full lossof the anti-cytophagal activity of tannic acid . Ofcourse, there are many aquacultural factors suchas temperature, pH, light, dissolved oxygen, totalalkalinity and hardness, other metal elements (ironand copper), and nutrients (e .g ., casein and vitaminC) in pond systems, which have significance andinfluence the availability of tannic acid and relatedcompounds (unpublished data) .Although gallic acid has relatively little binding

capacity towards protein and glycogen, our resultshave shown that gallic acid was superior to theother test compounds in bacterial toxicity towardsC. clumnaris and cyanobacteria (Chung et al .1995a) . This suggests that another mechanism fortannins' bacterial toxicity is a possibility . The 1-carboxyl group of gallic acid may become chargedby protonation or uncharged by deprotonation atphysiological pH . The uncharged species may ac-celerate both the rates of diffusion and the finalequilibrium distribution of the compound acrossthe membrane bilayer of some bacteria (Nikaidoand Thanassi 1993). It should also be noted thatthe various types of ester functionality on the tan-nin molecule are postulated to play a role in bac-terial toxicity . Our preliminary work showed thatthose compounds with alkyl groups strongly in-hibited the gliding motility of C. columnaris (datanot shown) . This suggests that action on the struc-tural elements of the bacterial membrane by esterlinkages may be relevant .

In general, it is concluded that a close relation-

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ship appears to exist between chemical structureand bacterial toxicity . The hydroxyl groups of tan-nins are considered to be essential for antibacterialactivity .

Regardless of the possible mechanisms of ac-tion, tannic acid and related compounds seem tobe strong inhibitors of the growth of C. columnaris.Further study, such as investigation of tannin con-centration in pond systems and its impact on fish,is necessary for assessing the commercial appli-cation potential of these compounds . In the naturalaquatic environment, tannins may be important in-digenous factors in the control of fish diseases .

AcknowledgmentThis research was supported by the endowment

of the W. Harry Feinstone Chair of Excellence inMolecular Biology held by S .E.S .

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