Synergistic Growth Studies of Entmoeba Gingivalis Using an Ecologen_Gannon

8/2/2019 Synergistic Growth Studies of Entmoeba Gingivalis Using an Ecologen_Gannon

1/5

lnrernnrional Journa lfor Parasit ology Vol. 22, No. 1, pp. 9277931, 1992Primed in Grral Brirom

002&7519/92 $5.00 + 0.00Pergomon Press Ltd

0 I992 Aurrralran Socieiy for Porosirology

SYNERGISTIC GROWTH STUDIES OF ENTAMOEBA GINGIVALISUSING AN ECOLOG ENR

JOHN T. GANNON and HARALD A. B. LINKE*Department of Microbiology, New York University Dental Center, 421 First Avenue, New York,

NY 10010, U.S.A.(Received 24 March 1992; accept ed 15 June 1992)

Abstract- GANNON J. T. and LINKE H. A. B. 1992. Synergistic growth studies of Entamoeba gingivalis usingan EcologenR. International Journal f or Parasitology 22: 927-93 I. A unique multiple diffusion growthchamber, an EcologenR, designed for the study of interactions among microorganisms, was introduced as ameans of growing xenic cultures of Entamoeba gingivalis with Crithidia sp. or Yersinia enterocolitica.Enfamoeba gingivalis was grown in the central diffusion reservoir of the EcologenR connected to separategrowth chambers inoculated with the microorganisms to be evaluated. Growth of the accompanyingbacteria in the E. gingivalis compartment was almost completely eliminated, except for sparse Pseudomonassp. growth. The most vital E. gingivalis cultures were observed when either Crithidia sp. or Y. enterocoliticawere added to the EcologenR 48 h prior to the E. gingivulis inoculum. The medium which provided the bestgrowth of the oral protozoan in this system was the new improved E. gingivaks medium containingantibiotics.INDEX KEY WORDS: Entamoeba gingivalis; xenic growth; protozoa; Crithidia; Yersinia enterocolitica.

INTRODUCTIONPREVIOUS studies (Boeck & Drbohlav, 1925; Diamond,1968, 1983; Gannon & Linke, 1989a; Howitt, 1925;Wantland, Wantland & Winquist, 1963) have reportedthe xenic cultivation of Entamoeba gingivalis. How-ever, the monoxenic and axenic cultivation of this oralprotozoan, which would be important for clinicalstudies (Linke, Gannon & Obin, 1989), has never beenachieved. Entamoeba gingivalis is difficult to maintainin virro, since the oral bacteria isolated with theamoebae are usually unfavorable for their growth(Diamond, 1983; Gannon & Linke, 1989b). A growthstudy using an EcologenR was conducted in order todetermine if symbionts (protozoa as well as bacteria),grown in separate compartments of this instrument,could enhance growth of E. gingivalis dwelling in amembrane-interconnected central diffusion reservoir.

MATERIALS AND METHODSQuantiration of E. gingivalisgrowfh. The quantitation of E.

gingivalis growth was based on the number of amoebaeobserved per field. For each sample, approximately one dropfrom a 22.8-cm Pasteur pipette (Fisher Sci., Springfield, NJ)was placed on a 75 x 25 mm glass slide (VWR Sci., SouthPlainfield, NJ), which was subsequently covered with a 22 x22mmcover-glass (no. I, thickness0.134.17 mm; VWR Sci.,

* To whom all correspondence should be addressed.

South Plainfield, NJ). Results were obtained by calculatingthe number of E. gingivalis observed per field after severalreadings using a phase-contrast microscope (American Op-tical, Buffalo, NY) at a magnification of 40 x (field of view0.36 mm).

Trophozoife activity. The same slides were observed asdescribed under Quantitation of E. gingivulis growth.However, only amoebae that w ere observed to exhibit truepseudopod movement or that underwent any visible changein cell morphology were counted. Trophozoite activity wasexpressed as per cent motile vs non-motile of total tropho-zoite count.

Ecologe# studies. An EcologenR (New Brunswick Scien-tific Co., Edison, NJ) is a multiple diffusion growth chamberdesigned for the study of interactions among micro-organisms. It consists of a stainless steel central diffusionreservoir C (Fig. I), surrounded crosswise by four cylindricalborosilicate glass growth chambers A and B (only two out offour chambers used; Fig. 1). The growth chambers and thecentral diffusion reservoir have a working capacity of lO(t300 ml and 500 ml, respectively. Both diffusion reservoir andgrowth chambers are equipped with two multipurpose ports,which are used for inoculation, addition of nutrients,sampling of the medium, and measurement of pH anddissolved oxygen. Membrane filters (0.45 pm) were installedbetween the central diffusion reservoir and the growthchambers, allowing interactions of cell metabolites and co-factors to be investigated during cultivation. The completeapparatus is repeatedly steam sterilizable. An EcologenR wasused to support the growth of E. gingivalis (L. S. Diamondsstrain HU-304:NIH, ATCC 30927, xenic) and its associatedmicroflora with either Yersinia enterocolitica, isolated from

927


2/5

928 J. T. GANNON and H. A. B. LINKETABLE I-MODIFIED TYSGM-9 MEDIUM OR TH E CULTIVATIONOF Entamoeba

gingivalis

Components source* AmountsPotassium phosphate, dibasic F 2.8 gl Potassium phosphate, monobasic F 0.4 gl~Tryptone D 2.0 gl_Yeast extract D 1.0 gl_Gastric mucin D 2.4 g 1~NaCl F 7.5 g I Distilled water 970.0 mlAfter sterilizing in an autoclave and cooling, the following were added:

Heat-inactivated bovine serum B 40.0 ml I Tween 80 solution s 1.0 mll Rice flour solution (0.5 g heat-sterilized rice flourdissolved in 9.5 ml sterile saline) D 0.2 ml/8 ml

pH adjusted to 7.2 with 1 N NaOH*Source: F, Fisher Scientific, Springfield, NJ; D, Difco, Detroit, MI; B,

Biofluids, Rockville, MD; S, Sigma, St Louis, MO.

FIG. 1. EcologenR, a multiple diffusion growth chamber forthe study of interactions among microorganisms, withcentral growth chamber C and four adjacent compartments

(only two chambers, A and B, were used).

the E. gingivalis stock culture (Gannon & Linke, 1989b) orCrifhidia sp. (L. S. Diamonds strain Ref-l:PRR, ATCC50083). The media used were TYSGM-9 medium (Diamond,1982), which was slightly modified (Table l), E. gingivalismedium containing antibiotics (Gannon & Linke, 1990)

summarized in Table 2, and antibiotic-free E. gingivalismedium (Gannon & Linke, 1991), as shown in Table 3. Togrowth chambers A and B (Fig. I), 250 ml of medium wasaseptically added, and 400 ml of medium was asepticallyadded to the central diffusion reservoir C (Fig. 1). Theantibiotics used were 125 pg mlpiperacillin (Lederle,Carolina, Puerto Rico), 25 jrg ml neomycin (MannResearch Lab., New York, NY), 1000 units ml- penicillin G(Sigma, St Louis, MO), and 75 pgrnl- erythromycin (Sigma,St Louis, MO). Entamoeba gingivalis with its accompanyingmicroflora was inoculated (12.5 ml) into growth chamber A.Growth chamber B was inoculated with either Y. enlero-colifica (25 ml), Crithidia sp. (25 ml) or a control (25 ml; nomicroorganisms added). The EcologenR was then mounted ina gyratory water-bath shaker (Model G76, New BrunswickScientific Co., Edison, NJ) and incubated at 34.5-35C with ashaker speed of 60 r.p.m. for 48 h. After incubation, theEcologenR was placed in an ice water-bath for 10 min. Bothgrowth chambers were aseptically decanted, leaving approx-imately 25 ml of medium. Wet-mount preparations of the E.gingivalis cultures were examined using a phase-contrastmicroscope to determine the numbers of Emamoeba presentand the extent of trophozoite activity. The amoeba culture ingrowth chamber A was agitated to loosen any amoebaeadhering to the walls of the chamber. The E. gingivalis cultureand the growth chamber B culture were then transferred withthe aid of a pipette into separate 50 ml centrifuge tubes,chilled in an ice water-bath for 5 min and then centrifuged at275 g for 3 min. The supernatants were decanted and thepellets were resuspended in 25 ml of sterile 0.85% salinesolution. All residual medium was then removed from thecentral diffusion reservoir C, and the Ecologens was sterilizedin an autoclave (30 min at 103.42 kPa). Fresh medium wasaseptically added, and the growth chambers were reinoc-ulated using the resuspended pellets from the precedingEcologens cultures. All growth studies were carried outin duplicate.


3/5

Entam oeba gi~gjl~ai~s kologen R studiesTABLE 2-Entamoebagingivalis MEDIUMCONTAININGANTIBIOTICS

929

Components*K,HPO,KH,PO,TryptoneYeast extractDistilled water

w 2.8 g NaCl F) 7.5 g(F) 0.4 g Gastric mucin (D) 2.4g(DI 2.0 g Ascorbic acid (St 0.2 g(D) 1.0 g Ferric ammonium (S) 228.0 mgcitrate

960.0 mlAfter sterilizing in an autoclave and cooling the following were added:Heat-inactivated bovine serum (B) 40.0 mlTween 80 solution (S) 0.5 mlRice starch solution (0.5 g heat-sterilized rice starch dissolvedin 9.5 ml sterile saline) (A) 0.1 ml/8 mlPiperacillin (L) 125.0 pg ml ErythromycinNeomycin (M) 20.0pgml PenicillinModified special 107 vitamin mix

Aseptically combine the following:Diamonds TPS- I vitamin solution (N),40 x diluted to I x concentration 100.0 mlSolution I: 40 mg vitamin B,z (S)

in 100 ml dist. H,O I .2 mlSolution 2: 50 g Tween 80 (S)

in 100 ml abs. ethanol 0.4 mlSterile distilled water 18.4 ml

(3 75.0flgml (3 1000.0 units ml 30.0 ml

* Source: F, Fisher Scientific, Springfield, NJ; D. Difco, Detroit, MI; B, Biofluids, Rockville, MD; S, Sigma, StLouis, MO; A, American Key Products, New York, NY: L, Lederle, Carolina, Puerto Rico; M, Mann Research Lab.,New York, NY: N, North American Biologicals, Miami, FL.

TABLE 3--ANTIBIOTIC-FREE niuin~eb~g~~gjval~~MEDIUMComponents*Tryptone (D) 2.5 g Ascorbic acid (S) 0.2 gYeast extract (D) 1.25g K,HPO, (F) I.0 gDextran (S) 2.0 g KH,PO, (F) 0.6gGastric mucin (D) 2.4 g Ferric ammonium citrate (S) 0.2 gNaCl (F) 2.0 e Distilled water 870.0 mlAfter sterilizing in an autoclave and cooling the following were added:

Heat-inactivated bovine serum (B) 100.0 mlModified special 107 vitamin mix 30.0 ml

Aseptically combine the following:Diamonds TPS-I vitamin solution (N),40 x diluted to I x concentration 100.0 mlSolution 1: 40 mg vitamin B,?(S)

in 100 ml dist. H,O 1.2 mlSolution 2: 50 g Tween 80 (S)

in 100 ml abs. ethanol 0.4 mlSterile distilled water 18.4 ml

pH adjusted to 6.8 with I N NaOH* Source: D, Difco, Detroit, MI; S, Sigma, St Louis, MO; F, Fisher Scientific. Springfield, NJ; B.

Biofluids. Rockville, MD; N, North American Biologicals, Miami. FL.


4/5

930 J. T. GANNOK and H. A. B. LIMETABLE 4-Entamoeba gingivalis ECOLOGEN~ TUDY

Culture E. gingivalis count Trophozoite activity*1a, I-b, I-C i++,+++++,++++ ++,++++,+++2-a. Z-b, 2-c +i. +i++, t-k+ +-I-++++, ++3-a, 3-b, 3-c -t-++, +++-I-, +++ ++,++++,+++4-a. 4-b, 4-c ++, +++, +++ ++, +++, ++5-a, 5-b, 5-c +, ++, ++ +, ++. ++

I, Crithiriia sp. added 48 h before E. gingivalis; 2, Crifhinia p. added at the same timeas E. ~j~~iva~is;3, Y. enteroco~iti~a dded 48 h before E. gi~~i~la[is; . Y. ~~reroeu[iti~~added at the same time as E. gj~g~va~js: , control (no organisms added): a, modifiedTYSGM-9 medium (see Table I): b, E. gingivalis medium with antibiotics (see Table2); c, E. gingivalis medium without antibiotics (see Table 3).

* Per cent of active E. Xingivalis trophozoites per field.E. gingivaks count

+ - I-IO/field++ - ll-15ifield

+++ -- 16-20 / field++++ -- 21-2S:lield

+ + + + + --- over 25 / field

Trophozoite activityup to 25%> 25% but i 50%> 50% but < 75%> 75% but < 100%100%

RESULTS AND DtSCUSSIONThe results of the Ecologen studies are summarized

in Table 4. Crithidia sp. was chosen as a possiblesymbiont because it had been used for the monoxeniccultivation of Erztumoeba histo@tica (Diamond, 1968).Yersiniu e~teroco~~tica was utilized because it wasisolated from the accompanying microflora of theoriginal xenic E. gingivalis stock culture (Cannon &Linke, 1989b). In comparison to controls, the use ofeither Y. enterocolitica or Crithidia sp. in growthchamber B resulted in an increase in both the numbersand trophozoite activity of E. gingivaalis in growthchamber A. These results indicated that a cellularmetabolite produced in growth chamber B diffusedacross to the E. gingivalis growth chamber causing abeneficial effect on E. gingivulis growth. Crithidia sp.was more effective than Y. enterocolitica in supportingE. ~~~~j~~~~~growth. Bacterial growth was barelydetectable (fewer than one bacterium observed perfield) in the E. gingivulis growth chamber. The fewaccompanying bacteria were identified as a Pseudomonussp. (detection on undiluted spread-plates). Althoughpolymyxin B would have been a suitable antibiotic forcontrolling this bacterium. as observed in anotherindependent experiment (data not shown), it was notpossible to completely eradicate the Pseudomonas sp.without also having a negative effect on Entamoehagrowth. Therefore, the amoeba culture used was amonoxenic culture (K gingivalis- Pseudomonas sp.)supported by either Crithidia sp. \r Y. cntcroc~ofiticu

which were grown in a separate growth chamberconnected by the diffusion reservoir. The controls(Table 4) indicated declining numbers and trophozoiteactivity of E. gingivulis. Therefore, it was necessary tohave the symbionts growing in the adjacent growthchamber B to iR]~rove growth of the oral protozoan.The Ecolog& was a very useful tool because bygrowing E. gingivulis and either Crithidiu sp. or Y.enterocolitica in separate growth chambers, there Wasno competition for nutrients. Therefore, all of thecompeting microorganisms were grown under optimalconditions. In other experiments where the symbiontwas grown in the same test-tube with the oral proto-zoan, a reduced growth and trophozoite activity of E.gingivalis was observed (data not shown). Anothersignificant observation was that E. gingivalis growththrived when the symbiont was added 48 h prior to theE. g~~g~~ff~~s ncubation. This provided additionalevidence that the symbiont conditioned the mediumto enhance E. gingiva1i.s growth. The E. gingivahsmedium containing antibiotics was slightly moreeffective in supporting growth of the oral protozoanthan the antibiotic-free E. gingivalis medium. Ingeneral. both media (Gannon & Linke, 1990. 1991)supported excellent growth of E. ging~vulis under theseconditions..4f~l;noM/f,~~c,~~CNI.C--Thls esearch was in part supported bya grant from the National Institute of Dental Research (DSRI R03 DE07015-01).


5/5

Entamoeba gingivalis EcologenR studies 931REFERENCES

BOECK W. C. & DRBOHLAV . 1925. The cultivation of EM-am oeba histo lyt ica. A merican Journal of Hy giene 5: 311407.

DIAMOND L. S. 1968. Improved method for the monoxeniccultivation of Enfamoeba histolytica Schaudinn, 1903 andE. histolytica-like amoebae with trypanosomatids. Journalof Parasitolo gy 54: 7 15-I 19.

DIAMOND L. S. 1982. A new liquid medium for the xeniccultivation of Entamoeba histolytica and other lumendwelling protozoa. Journal of Parasi to logy 68: 958-959.

DIAMOND L. S. 1983. Lumen dwelling protozoa: Entamoeba,trichomonads and Giardia. In: In-vitro Cultivarion ofProtozoan Parasites (Edited by JENSEN J. B.), p. 76. CRCPress, Boca Raton, FL, U.S.A.

GANNON J. T. & LINKE H. A. B. 1989a. Growth studies onxenic cultures of Entamoeba gingivalis using establishedmedia. Internationa l Journal,for Parasitolo gy 19: 835-838.

GANNON J. T. & LINKE H. A. B. 1989b. Studies on the

microflora associated with xenic cultures of Entamoebagingival is. Microbios 58: 95-100.

GANNON J. T. & LINKE H. A. B. 1990. new mediumcontaining antibiotics for the xenic cultivation ofEntam oeba gingivali s. Parasitology Research 76: 643-641.

GANNON J. T. & LINKE H. A. B. 1991. An antibiotic-freemedium for the xenic cultivation of Entamoeba gingivalis.Internationa l Journalfor Parasitol ogy 21: 403407.HOWITT B. F. 1925. The cultivation of Entamoeba gingivalis(Gros). Univ ersity qf Cal[fornia Publicat ions in Zoology 28:66-122.

LINKE H. A. B., GANNON J. T. & OBIN J. 1989. Clinical surveyof Entamoeba gingivalis by multiple sampling in patientswith advanced periodontal disease. International Journalfor Parasi to logy 19: 803-808.

WANTLANDW. W., WANTLANDE. M. & WINQUISTD. L. 1963.Collection, identification and cultivation of oral protozoa.Journal qf Dent al R esearch 42: 1234-1241,

Documents

Synergistic Growth Studies of Entmoeba Gingivalis Using an Ecologen_Gannon