INTERNATIONAL JOURNAL OF LEPROSY^ Volume 57, Number 3

Printed in the U.S.A.

Susceptibilities of Mycobacterium leprae and M. aviumComplex to the H 202-Fe-mediated Halogenation

System Supplemented with Antimicrobial Agents'Haruaki Tomioka, Yoshitaka Yamada, Hajime Saito, and Joji Jidoi 2

The H 20 7 -Fe-mediated halogenation sys-tem has a potent antimicrobial activity(4,9 ' 10) against intracellular parasites, in-cluding mycobacteria ( 13), and it is thoughtto play an important role in the expressionof antimicrobial activity of phagocytes. My-cobacterium leprae, an intracellular para-site, resists the bactericidal mechanisms ofphagocytes. It is impossible to estimate thenumber of live organisms of M. leprae bycolony counting, because Al. leprae cannotbe grown in vitro on any known media. Ithas been reported that fluorescein diacetate-ethidium bromide (FDA/EB) staining isuseful for determining the viability of my-cobacterial cells, including Al. leprae ( 12).This method is based on the following prin-ciple: FDA, a nonpolar and nonfluorescentfatty-acid ester, is transferred into intact cellsthrough the cell membrane by an activetransport system and is then hydrolyzed bycytoplasmic esterases, yielding polar flu-orescent fluorescein which emits green lightin response to excitation by ultraviolet light.Thus, viable cells are stained green. Ethid-ium bromide, on the other hand, can easilypenetrate into dead cells through the dam-aged cell membrane but it is excluded byviable cells. EB is tightly bound to double-stranded DNA. This results in an orangefluorescence of dead cells (2. ").

We examined the susceptibility of AI. lep-rae and a member of the M. avium complex(MAC) to the microbicidal activity of thehalogenation system, with or without anti-mocrobial agents, using FDA/EB staining.

' Received for publication on 21 March 1989; ac-cepted for publication on 24 April 1989.

2 H. Tomioka, Ph.D.; H. Saito, M.D., Ph.D., De-partment of Microbiology and Immunology; Y. Ya-mada, M.D., Ph.D.; J. Jidoi, M.D., Ph.D., Departmentof Dermatology, Shimane Medical University, Izumo693, Japan.

MATERIALS AND METHODSOrganisms. Al. leprae Thai 53 was har-

vested from the foot pad tissue of nude miceinfected with Al. leprae obtained from K.Kohsaka, Research Institute for MicrobialDiseases, Osaka University, Osaka, Japan.The M. /eprae-infected foot pad tissue wasminced with scissors and homogenized witha glass homogenizer in Hanks' balanced saltsolution (HBSS) with 5% fetal bovine serum(FBS). This homogenate was centrifuged at100 x g x 5 min and four fifths of thesupernate was again centrifuged at 400 x gx 20 min. The pellet was suspended in HBSSwith 5% FBS and centrifuged at 100 x g x5 min. Three fifths of the supernate wasdiluted with 0.15 M saline to the appropri-ate concentration. The strain of the M. av-ium complex, ATCC 13950, supplied by A.Y. Tsang (National Jewish Hospital and Re-search Center, Denver, Colorado, U.S.A.)was cultivated in Dubos' Tween () -albuminmedium (Eiken Chemical Co., Tokyo, Ja-pan) at 37°C for 7 days. The organisms wereharvested by centrifugation (500 x g x 15min), washed twice with 0.15 M saline, andresuspended in saline.

Chemicals. Fluorescein diacetate (FDA)and ethidium bromide (EB) were purchasedfrom Sigma Chemical Co., St. Louis, Mis-souri, U.S.A. The antimicrobial agents usedwere: isonicotinic acid hydrazide (isoniazid,INH) (Wako Pure Chemical Co., Osaka, Ja-pan); p-amino-salicylic acid (PAS) (Wako);4,4'-diaminodiphenylsulfone (dapsone,DDS) (Nakarai Chemical Co., Kyoto, Ja-pan); rifampin (RFP) (Daiichi Pharmaceut-ical Co., Tokyo, Japan); ofloxacin (OFLX)(Daiichi); cefoxitin (CFX) (Daiichi); clofa-zimine (CLO) (CIBA-GEIGY Co., Hyogo,Japan); and cofetatan (CTT) (YamanouchiPharmaceutical Co., Tokyo, Japan).

Antimycobacterial activity of the halo-genation system. The microbicidal activity

628

57, 3^Tomioka, et al.: H202-Fe-nzediated Halogenation^629

TABLE 1. Effect of the halogenation sys-tem, with or without antileprosy agents, onthe intactness of M. leprae determined on abasis of FDA/EB staining."

Substrates"(11M)

Additions'(100 AM)

Percentage ofgreen-stained cellsd

0 - 50.5 ± 2.1100 - 50.0 ± 1.5

0 RFP 38.4 ± 2.0100 RFP 40.1 ± 2.5

0 DDS 53.4 ± 3.2100 DDS 33.8 ± 0.8

0 CLO 44.0 ± 3.7100 CLO 11.0 ±^1.0

The organisms were treated by the halogenationsystem with or without indicated agents at 37°C for 60min and stained with FDA/EB to determine the per-centage of the green-stained cells as a parameter ofintact cells.

" The halogenation system consists of various con-centrations of substrates (sodium iodide, hydrogen per-oxide, ferrous sulfate) and 20 mM acetate buffer (pH5.5).

RFP = rifampin; DDS = dapsone; CLO = clofa-zi m inc.

Mean ± S.E.M. (two separate incubations).

of the halogenation system was measuredas described previously ( 13). Briefly, the re-action mixture (2 ml), consisting of 100 AMsodium iodide, 100 AM hydrogen peroxide,100 ALM ferrous sulfate, various concentra-tions of antimicrobial agents, 20 mM ace-tate buffer (pH 5.5) and test organisms (ata final concentration of 1 x 10 7) organisms/ml), was incubated in a shaking water bathat 37°C for 60 min. The viability of the M.leprae in the resulting incubation mixturewas determined by FDA/EB staining. In thecase of the M. avium complex strain, thenumber of residual colony-forming units(CFU) in the incubation mixture was esti-mated by plating on Middlebrook 7H 10 agarplates (Difco Laboratories, Detroit, Mich-igan, U.S.A.).

FDA/EB staining. FDA/EB staining wasdone by the method ofTsukiyama (I 7 ). Brief-ly, the reaction mixture was diluted with 4ml of phosphate buffered saline (PBS) (pH7.2) after incubation and centrifuged at 1000x g x 30 min. The organisms were sus-pended in 0.1 ml of Dubos' Tween s -al-bumin medium and smeared on a glass slide.The FDA/EB solution consisted of 0.5 mlof 1 mg/ml FDA in acetone, 0.02 ml of 2mg/ml EB in PBS (pH 6.5) containing 0.05%

TABLE 2. Effect of the halogenation sys-tem, with or without antimycobacterialagents, on the intactness of M. leprae deter-mined on a basis of FDA/EB staining.a

Substrates^Additions"^Percentage of(pM)^(100 AM)^green-stained cells

0 - 50.8 ± 0.8100 - 52.4 ± 4.2

0 PAS 51.5 ± 0.1100 PAS 53.1 ± 4.9

0 INH 48.9 ± 1.9100 INH 52.1 ± 1.7

See footnotes to Table 1.'' PAS = p-amino-salicylic acid; INH = isonicotinic

acid hydrazide.

Tween® 80 (PBS-Tween® 80) and 4.5 mlof PBS-Tween® 80. After the smear hadbeen incubated in the FDA/EB solution at37°C for 60 min, the numbers of green- andorange-stained cells were counted at a mag-nification of 400 under a fluorescent micro-scope. The percentage of green-stained cellsin the total cells (green- plus orange-stainedcells) was then calculated.

RESULTSSusceptibility of M. leprae to the halo-

genation system with or without antimicro-bial agents. Table 1 shows the changes inthe viability of Al. leprae exposed to thehalogenation system, with or without theaddition of antileprosy agents, as deter-mined by FDA/EB staining. The percentageof green-stained cells was not affected by thehalogenation system alone but was reducedby the system combined with either RFP,DDS, or CLO. It is noted that RFP alonereduced the viability of the organisms. Thesynergistic effect was highest with CLO, fol-lowed by DDS. In cases of the antituber-culosis drugs (Table 2) such as PAS andINH, there was no such decrease. As shownin Table 3, neither CFX nor CTT caused asignificant decrease in green-stained cells,even in combination with the halogenationsystem; the halogenation system plus OFLXcaused a marked reduction in the viabilityof the organisms.

Susceptibility of M. avium complex to thehalogenation system. Table 4 shows the sus-ceptibility of the MAC strain to the halo-genation system on the basis of changes inviable units and in the ratio ofgreen-stained

630^ International Journal of Leprosy^ 1989

TABLE 3. Elli,ct of the halogenation sys-tem, with or without antibacterial agents, onthe intactness of M. leprae, determined on abasis of FDA/ER staining."

Substrates^Additions"^Percentage of(PM)^

(100 pM)^green-stained cells

0 - 55.2 ± 0.2100 52.2 ± 1.5

CFX 53.6 ± 2.2100 CFX 54.1 ± 0.6

0 CTT 52.8 ±^1.1100 CTT 54.0 ± 2.5

0 OFLX 55.6 ± 3.1100 OFLX 35.9 ±^1.8

See footnotes to Table 1.CFX = cefoxitin; CTT = cefotetan; OFLX = of-

loxacin.

cells after FDA/E13 staining. The halogena-tion system did not affect the behavior ofthe organisms in FDA/EB staining, butmarkedly reduced the number of residualCFU. This indicates that, in the case of theMAC strain, the ability of the organisms tobe stained green by FDA/EB did not cor-relate with the ability to grow on 7H 10 agarmedium. Table 5 shows the effects of thehalogenation system supplemented withOFLX on the green-fluorescence intensityof the MAC strain. Even in the presence ofOFLX, the halogenation system failed toreduce the number of the green-stained or-ganisms by FDA/E13 staining.

TABLE 4. Relationship between changesin membrane intactness determined by FDA/EB staining and reduction in viable units bytreatment of a MAC strain with the halo-genation system."

Substrates(AM)^green-stained

Percentage of

cells^log CFU/ml

0 98.7 ±^1.3 7.13 ± 0.2710 97.3 ±^1.5 7.48 ± 0.0750 97.5 ± 0 6.43 ± 0.15

100 96.7 ±^1.3 0"

The organisms were incubated in the halogenationsystem consisting of various concentrations of sub-strates (hydrogen peroxide, ferrous sulfate and sodiumiodide) and 20 mM acetate buffer (pH 5.5) at 37°C for60 min, and subjected to FDA/E13 staining and CFU-counting on 7H10 agar plates. Other details are as inTable 1.

"

TABLE 5. E,fJect of the halogenation sys-tem, with or without olloxacin, on the in-tactness of an M. avium complex strain de-termined on a basis of PDA/EB staining."

Substrates(PM)

011oxacin(pg/ml)

Percentage ofgreen-stained cells

0 0 97.2 ± 0.90 100 99.2 ± 0.3

100 0 98.6 ±^1.0100 25 99.2 ± 0.1100 100 100.0 ± 0

See footnotes to Table 1.

DISCUSSIONThe peroxidase- or Fe-mediated haloge-

nation system has potent bactericidal activ-ity (4.9. 10. "). This antimicrobial mecha-nism seems to be mediated by iodinationand oxidation of cellular components req-uisite for viability, such as membrane-pro-teins and iron sulfur centers of the electrontransport system (4.9. 10). FDA is taken upby cells due to an active transport systemof the cell membrane and subsequently ishydrolyzed by intracellular esterases, givinga green fluorescence. EB enters cells throughthe damaged cell membrane and producesan orange fluorescence by binding to DNA.These events lead to a reduction of greenfluorescence and an increase in orangephotocmission in the case of a damaged cellmembrane (t 2). Here, we found that theH,0,-Fe-mediated halogenation systemalone did not reduce the green-fluorescenceintensity of M. leprae or the MAC strain.Therefore, it seems that the halogenationsystem alone failed to cause damage to thecell membrane of M. leprae, at least not toa degree that resulted in the reduction ofits green fluorescence.

On the other hand, when the halogenationsystem was supplemented with OFLX, so-treated M. leprae showed a decrease in thenumber of green-stained cells in FDA/EBstaining. OFLX primarily inhibits the su-percoiling activity of DNA gyrase, and thusit does not directly cause any cell membranedamage ( 3). Therefore, it is thought that thehalogenation system, even combined withOFLX, could not damage the cell mem-brane structure. However, it is still possiblethat the antimicrobial system might inac-tivate the active transport system of the cell

57, 3^Totnioka, et al.: H20,-Fe-mediated Halogenation^631

membrane in an indirect manner, causingreduction of the green fluorescence of treat-ed organisms under FDA/EB staining.

DDS and CLO, which do not have a pri-mary action of the cell membrane (l. 7 ' 8),

also caused a decrease in the number ofgreen-stained Al. leprae when used in com-bination with the halogenation system. Themechanisms of the cell membrane function-disintegration action of these agents may besimilar to that of OFLX. RFP alone reducedthe percentage of green-stained cells. How-ever, RFP is known to primarily inhibit theRNA polymerase of organisms ( 6). In stud-ies using an electron microscope, damage tothe structure of the cytoplasm, ribosomesand mcsosome of the organisms by RFPwas observed, but structures of the nucleus,cytoplasmic membrane and cell wall werepreserved ( 5). Thus, while RFP does notcause structural damage to the cell mem-brane, it is possible that the integrity of thecell membrane of the organism might bereduced when protein synthesis is inhibitedby RFP.

In the case of the MAC strain, the numberof viable units was decreased by the halo-genation system but the green-fluroescenceintensity of the organisms in FDA/EB stain-ing was not affected, even when the halo-genation system was combined with OFLX.This implies that the halogenation systemmay inactive certain cell components es-sential for growth of the organisms withoutdamaging the active transport activity or theintegrity of the cell membrane. These resultssuggest that the functional state of the Al.leprae cell membrane is much more sus-ceptible to damaging action of the halogena-tion system supplemented with OFLX thanis the MAC strain cell membrane. If suchis the case, the structural and functional sta-bility of the M. leprae cell membrane, in-cluding the active transport system, isthought to be much less than that of theMAC strain cell membrane.

In any case, this study revealed that areduction of the green fluorescence of Al.leprae cells in FDA/EB staining was pro-duced when Al. leprae cells were treated withthe Fe-mediated halogenation system com-bined with an anti-Al. leprae agent. Thissytem may be useful for in vitro screeningof antileprosy agents.

SUMMARYThe susceptibilities of Alycobacteriunz

leprae and Al. avizenz complex (MAC) to theH,-0,-Fe-mediated halogenation systemsupplemented with antimicrobial agentswere evaluated by fluorescein diacetate-ethidium bromide (FDA/EB) staining. Inthe case of Al. leprae, the number of green-stained bacteria (intact cells) was reducedin the presence of the H20,-Fe-mediatedhalogenation system supplemented withagents possessing antileprosy activity, suchas rifampin, 4,4'-diaminodiphenylsulfone(dapsone), clofazimine, and ofloxacin. In thecase of the MAC strain, although viable unitsof the organisms were reduced by the halo-genation system alone, the number of green-stained cells in the FDA/EB stain was notreduced, even when the halogenation sys-tem was used in combination with ofloxa-cin. Because stainability of the cells is re-lated to structural and functional intactnessof the membrane, differences between M.leprae and the MAC strain imply possibledifferences in the rigidity of the cell mem-brane.

RESUMENUtilizando la tinciOn con diacetato de fluoresceina-

bromuro de etidio (DAF/BE) se evaluO la susceptibi-lidad del Mycobacterium leprae y del complejo del M.(alum (MAC) a la halogenaciOn mcdiada por el sistcmaH 20,-Fe suplementado con agentes antimicrobianos.En cl caso del M. leprae, el nUmero dc bactcrias tenidasde verde (celulas intactas) se rejudo en presencia delsistcma de halogenaciOn mediado por el F1,0 2-Fe su-plementado con agentes antileprosos tales como rifam-pina, 4, 4'-diaminodifensilsulfona (dapsona), clofazimi-na y ofloxacina. En el caso del complejo MAC, aunquelas unidades viables de los organismos disminuyeronen presencia solo del sistcma de halogenaciem, el nU-mero de celulas tenidas de verde (tinciOn DAF/BE) nose redujo ni siquicra cuando el sistcma de halogenaciOnse use en combinaciOn con la ofloxacina. Debido a quelas caracteristicas tintoriales de las celulas estãn rela-cionadas con la integridad estructural y funcional dela membrana, las diferencias entre M. leprae y MAC,implican posibles diferencias cn la rigidez de la mem-brana celular.

RESUME

On a evalue par une methode de coloration par lediacêtate-ethidium bromure de fluoresceine (FDA-EB),les susceptibilites respectives de Mycobacterium lepraeet du complexe Af. avium (MAC), au systeme d'halo-genation par peroxyde d'ox■,:gene et ter interposes, au-

632^ International Journal of Leprosy^ 1989

clue' on avait ajoute des agents microbicides. Dans le

cas de M. leprae, le nombre de bacterics colorees en

vert (c'est-a-dire de cellules intaetes), etait reduit enpresence du système d'halogénation decrit cidessus,

lorsqu'on y avait ajoute des agents ayant une activite

anti-lepreuse, lets que la rifampine, la 4,4',-diamino-

di phenyl-sulfone (dapsone), Ia clofazimine, et rolloxa-eine. Dans Ic cas du complexe de MAC, malgre le fait

que Ic nombre d'unites viables de ces organisms etait

reduit par Ic systeme d'halogenation utilise seul, le

nombre de cellules colorees en vent par Ic colorant

FDA/Ell n'etait pas diminue, et cc en &pit du fait que

le systeme d'halogênation etait utilise en combinaisonavec roflaxacine. La colorabilite des cellules &tam in-

flurencee par rintegrité structurelle et fonctionnelle deIa membrane, Ics diflirences notees entre M. leprae etIc complexe de .I/. minor dans cette etude suggerentque la rigidite des membranes cellulaires de ces orga-

nismes pourrait presenter des differences.

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