INTERNATIONAL. JOURNAL OF LEPROSY^ Volume 64, Number 3Printed in the U.S.A.

(ISSN 0145-916X)

Microbial Colonizers in Leprosy Skin Ulcers andIntensity of Inflammation'

A. Willem Sturm, Bushra Jamil, Keith P. W. J. McAdam, Khurram Z. Khan,Shaheen Parveen, Thomas Chiang, and Rabia Hussain 2

IVIrcobacterium leprae is the causativeagent of leprosy, a disease in which theclinical presentation varies with the pa-tient's immune response (' 5 ). Skin lesionsare seen at the lepromatous as well as thetuberculoid poles of the disease spectrum.Although the underlying immune pathologyof the lesions is different across the spec-trum, chronic ulceration can develop atboth poles of leprosy ("). These ulcers be-come infected with bacteria other than M.hp/we and show recurrent episodes of cel-lulitis of the surrounding tissues as well asosteitis of underlying bone. This, in turn, re-sults in deformities which disable patientsand contribute to the stigma associated withthe disease ( 1 ). Early and adequate treat-ment of these episodes of inflammationcould interrupt this sequence of events.

There are several reports (") whichshow that chronic skin ulcers in leprosy pa-tients are colonized with gram-positive rodswhich have not been observed in chronicskin ulcers of different etiology. However,there are no studies which compare cultureresults of leprosy-related and other skin ul-cers in the same population. Also, no at-tempt has been made to relate colonizingspecies with inflammation.

' Received for publication on 24 October 1995; ac-cepted for publication in revised form on 12 March1996.

= A. W. Sturm, M.D., Ph.D.; B. Jamil, N1.11.13.S.; K.Z. Khan. M.Sc., D.Bact.; S. Parveen, B.Sc., NI.Sc.; R.Hussain. B.Sc. (lions). M.Sc., Ph.D., M.R.C. (Path),Department of Microbiology, The Aga Kahn Univer-sity, Karachi, Pakistan. K. P. W. J. MacAdam, M.A.(Nat.Sc.), M.R.C.P., F.R.C.P., London School of Hy-giene and Tropical Medicine. London, U.K. T. Chiang,M.13.13.S., M.Sc., C.T.M., Marie Adelaide LeprosyCenter. Karachi. Pakistan.

Reprint requests to Prof. A. W. Sturm. Departmentof Microbiology, Medical School, University of Natal,P. 0. Box 17039, Congella 4013, South Africa.

This study compares organisms presentin leprosy skin ulcers with those present inskin ulcers of different etiologies, and cor-relates this with the severity of inflamma-tion. Susceptibility patterns of these organ-isms are reported and the possible contribu-tion of antimicrobial treatment towardprevention of deformity is discussed.

PATIENTS AND METHODSPatients were recruited from the popula-

tion attending Marie Adelaide LeprosyCentre (MALC) in Karachi, Pakistan. Allpatients with ulcers, from whom informedconsent was obtained in the period betweenOctober 1991 and October 1992, were en-rolled. Patients with nonleprosy skin ulcersof varying durations and not on antimicro-bial treatment for at least 2 weeks were re-cruited as a control group at the surgeryclinic of Civil Hospital, Karachi.

Inflammatory scores (IS) were measuredbased on the method of Ehrenkranz, et al.( 7 ). This included amount and character ofdrainage, extent of cellulitis, presence offoul odor and presence of necrosis. Obser-vations were scored as follows: drainage ondressing = 0; minimal, moderate and co-pious drainage in ulcer bed = 1, 2 and 4, re-spectively; serous, sanguinous, sanguino-purulent and purulent drainage = 0, 1, 3 and4, respectively; widest cellulitis width of< 1 cm, > 1 to 2 cm, > 2 to < 3 cm, > 3 to 4 cm = 0, I, 2, 3 and 4, respec-tively; no foul odor = 0; foul odor = 1; nonecrosis = 0; necrosis in ulcer margin = 1;necrosis in ulcer margin and base = 2. The ISvalue is the sum of these scores and rangesfrom 0 to 15.

Ulcers were sampled by slightly modify-ing the irrigation-aspiration method ( 7 ). Inbrief, at least 1 ml of sterile 0.85% sodiumchloride without preservatives is irrigatedunder the margin of the ulcer with a needle-less 5-ml syringe. Excess fluid is removed

274

64, 3^.S'turin, et al.: Microbial Flora of Leprosy Ulcers^275

TABLE I.^Characteristics of skin ulcers in patients with and without leprosy.

Leprosy patients (N = 55) Control patients (N = 18)

NUE ratio 40/15 15/3Mean (range) duration of ulcer (mos.) 10.3 (0.1-180) 7.5 (0.5-72)Character of ulcer (S/I) ratio)' 28/27 12/6Location and no. of ulcers

/lands 4 3Arms 3 1Feet 46 10 (p = 0.02)Legs 2_ 3Other 0 1

Mean no. of microbial species/sample 3.5 4.2

S/I) ratio = Superficial/deep ulcer ratio.

with a sterile gauze pad. The ulcer marginsare massaged circumferentially with a ster-ile cotton swab. After this, a second irriga-tion is performed using I ml of saline and anew syringe. Without removing the excessfluid, the ulcer margin is massaged again af-ter which the ulcer base and margins areswabbed with a sterile cotton-tipped swabwhich is immediately wrung into I ml ofbrain-heart infusion (BHI) broth for stain-ing and culture.

Smears of broth aspirate suspensionswere stained by means of Gram as well asKinyoun stain. Cultures were performed ona 7% sheep blood agar, cysteine lactoseelectrolyte-deficient medium (CLED), anisovitalex enriched chocolate blood agarand a nalidixic acid (10 mg/I) mecillinam (1mg/I) bilayered 7% sheep blood agar. Allplates were incubated at 37°C; the latter twoin a CO, incubator. In addition, a Sabouraudagar with 10 mg/I gentamicin and a Colum-bia agar with 2% Tween 80 and nalidixicacid 10 mg/1 were inoculated and incubatedat 30°C. Two sets of Lowenstein-Jensenslants incubated for 12 weeks at 30°C and37°C, respectively, were used for the cul-ture of mycobacteria. The presence ofanaerobes was evaluated on microscopy ofGram-stained smears, based on multiplicityof morphological forms, intracellular loca-tion, pale staining of gram negatives andsize as well as characteristic appearance.

Identification of organisms was done bymeans of established methods ("I) andbased on the latest edition of Berger's Man-ual of Deteminative Bacteriology

Minimal inhibitory concentrations (MIC)for antimicrobial agents were measured by

means of the agar dilution method on iso-sensitest agar with an inoculum of 10 1cfu/spot ( 11 ).

Mycobacterial culture media were ob-tained from DIFC0 Laboratories, Detroit,Michigan, U.S.A.; all others were obtainedfrom Oxoid, Basingstoke, Hampshire, En-gland.

Fisher's exact test was used to determinethe significance of differences between lep-rosy and control ulcers. Mann-Whitney andKendall rank order correlation coefficienttests were used for comparison/correlationbetween groups.

RESULTSFifty-five patients with leprosy skin ul-

cers and 18 control patients were enrolledin the study. Five of the study patients hadthe tuberculoid form of leprosy, 13 the lep-romatous and 37 had borderline leprosy.Thirty-nine patients, from whom a reliablehistory could be taken, had suffered fromthe disease for many years (mean duration14.2 years). At the time of enrollment. 30 pa-tients were not on any treatment, 10 were ondapsone, I was on clofazimine, and 14 wereon the multidrug therapy (MDT) regimen ofclofazimine, dapsone and rifampin. Onlythree patients had never been on anti leprosy ,medications; all the others had been treatedpreviously with different regimens.

Ulcer characteristics. Table I com-pares the characteristics of the skin ulcersof leprosy patients with those of the controlgroup. In leprosy patients, ulcers were pres-ent predominantly on the feet comparedwith the nonleprosy patients. A higher per-centage of leprosy ulcers were deep and of

276^ International Journal of Leprosy^ 1996

TABLE 2. Microbial species grown from skin ulcers of 55 leprosy patients and 18 non-leprosy patients.

Leprosy patients (N = 55) Control patients (N = I K)

Staphylococcus aureus 30 6Coagulase-negative staphylococci 19 12Streptococcu.s . pvogenes (1311S group A) 16 3Beta-hemolytic streptococcus 19 0

Group G (1311S group G)'Other streptococci 10 6Enterococcus species' K 7Arcanobacterium haemolytictme 10 0Unidentified branching rods' 7 0Corynebacterium species 40 9Bacillus species' 2 9Proteus mirabilis 7 4Other Etiterobacteriacene 8 5Pseduomonas aeruginosa 7 2Other Pseudomonas 9 6Acinewbacter calcoaceticus 4 1Other gram-negative rods 5 5Conduit, species 0 2Total 201 72

Present only in leprosy ulcers.h Present in significantly higher number (p = 0.04) in control ulcers.Present in significantly higher number (p = 0.03) in control ulcers.

longer duration than the control ulcers.Seven of the 18 nonleprosy patients suf-fered from post-traumatic ulcers, 6 had un-derlying diabetes mellitus, and 1 ulcer fol-lowed an insect bite; definite etiology couldnot be confirmed in 4 cases.

Ulcer microflora. When the microbialflora was compared in the study and controlgroups, no difference was found in the meannumber of microbial species per sample be-tween the two groups (Table 1). Table 2shows that leprosy patients had significantlymore beta-hemolytic streptococci. Group Gstreptococcus was exclusively present in ul-cers of leprosy patients (9.7% of isolates).Gram-positive rods accounted for 29.3% ofall isolates from the ulcers of leprosy pa-tients and 25% in the control patients. Thedistribution of species from the generaCorynebacteriutn and Bacillus was differ-ent in the two patient groups. In the leprosygroup, corynebacteria and branching rodsaccounted for 97% of gram-positive bacilliand Bacillus species constituted only 3% ofthe group. In the control patient group,Bacillus species formed 50% of gram-posi-tive rods; the rest were corynebacteria (p =0.03). In the leprosy group, one third of thegram-positive bacilli were branching rods.None of these were acid fast. Ten strains

were identified as Arcanobacteritan haemo-lytic:um; the remaining 7 could not he iden-tified with the methods used.

Skin ulcers of patients in the controlgroup grew yeast (two samples) and highernumbers of species of enterococci (p =0.04) and Enterobacteriaceae (p = 0.08)compared to the leprosy group. Moreover,no branching rods were isolated from thecontrol ulcers. Anaerobes (on microscopy)were found with the same frequency in thestudy (18%) and in the control (17%)groups. No mycobacteria could be culturedin either group.

Patients with leprosy had an inflamma-tory score (IS) which varied between 0 to13 (median of 2; mean score of 3). This wassignificantly (p = < 0.001) lower than in thecontrol group which showed a variation inIS between I and 10 (mean 5.5; median 6).No correlations were found between the ISand age of the patients, duration of ulcers,or type and duration of leprosy. The fourcontrol group ulcers, which etiology couldnot be ascertained, had a median IS of 1.5with a mean of 3.25 (range 1-9); this valueis similar to that in the leprosy group.

In the leprosy group, the number of spe-cies per sample appeared to be directly re-kited to the IS (Fig. I); no such correlation

14

2

8

6

4

2EE5t.1E.

2

0

-2

64, 3^Sturm, et al.: Microbial Flora of Leprosy Ulcers^277

0

0

0

o 00

OO

O 0 0 oO o

Q 0 0 0 0 0 00 0 0 0 0 0 0

O •

-1^0^1^2^3^4^5^6^7^8^9

NO. OF ISOLATES / ULCER

Ito. I. higher inflammatory scores appear to beassociated with higher numbers of isolates from thesample.

0

00

00O O

0

278^ International Journal of Leprosy^ 1996

TABLE 3. Susceptibility (MIC in pg/m1) of 20/ bacterial strains isolated from skinulcers of 55 leprosy patients and seven strains from skin ulcer.s . of 18 control patients.

Drug' RangeMean MIC MIC,„ MIC

, o,,Leprosy Controls Leprosy Controls Leprosy Controls

P 0.25-64 1.1 1.1 64 >64 >64 >64TRP 0.25-64 34 37.22 32 64 >64 >64OFX 0.001-2 0.3 0.4 0.125 0.25 0.5 IC 0.25-64 19.6 38.4 8 32 64 64

P= Penicillin, A= ampicil lin, CLOX = cloxacillin, CTX = celotaxime, R = rifampin, F = erythromycin, SMX= sulfamethoxazole, TMP = trimethoprim, OFX = ofloxacin, C = chloramphenicol.

piing technique used in this study has beenshown to he in decubitus ulcers as represen-tative for culture as a biopsy ( 7 ).

We observed that the microflora of skinulcers in leprosy patients was different fromthe flora of nonleprosy skin ulcers. Bothleprosy and control patients were from sim-ilar socioeconomic backgrounds and envi-ronmental differences are, therefore, un-likely to be accountable for those differ-ences. The significant observation was theisolation of gram-positive branching bacilliin leprosy ulcers, more than half of whichwere identified as A. haemolyticum. Thisorganism has not been reported previouslyfrom leprosy ulcers. It has been describedas a case report in relation to a post-trau-matic skin ulcer and as a causative agent ofoutbreaks of tonsillo pharyngitis ( 9). GroupG beta-hemolytic streptococci also were ex-clusively isolated from leprosy ulcers. Cer-tain species from the genus Corynebac-terium have been described as unique forleprosy patients ('). The branching rodsfrom our study patients which remainedunidentified could be similar to these lep-rosy-derived corynebacteria. Cell-wall com-position and DNA analysis of these strainsmay provide the answer ( 2 . ").

It has been postulated that the presence ofcertain grain-positive rods in leprosy ulcersis explained by their antigenic relatednessto M. leprae "). Tolerance to commonantigens perhaps allows these organisms tocolonize the leprosy ulcers. This would im-ply that all of the isolates which are unique

to leprosy ulcers may share some antigenicfeatures with M. leprae. At least two differ-ent organisms have been reported with thischaracteristic C• 13 ) hut neither one has beenclassified officially. From the present studywe may postulate that A. haemolvticwn,unidentified branching rods, and group Gstreptococci probably also belong to thisgroup. One would expect differences in col-onization with these organisms at the polesof the spectrum of leprosy depending on thepatient's immune status. We could not es-tablish such a relationship because of thesmall numbers of patients with tuberculoidand lepromatous leprosy. To resolve the is-sue of shared antigens, a comparative studybetween these organisms and M. leprae willbe required.

Isolation of group G streptococci fromleprosy patients only could reflect cross-infection in the health care facility fromwhich the patients were recruited. The samemay be true for A. haemolyticum and otherbranching rods. In such a situation, therewould be a clustering of cases in time.However, these organisms were isolatedthroughout the year. Also, if spread frompatient to patient or from health care workerto patient was a major factor, one would ex-pect to find the more virulent S. (litmus andgroup A streptococci. Hence, nosocomialoutbreaks cannot be held responsible forour observations.

Another possible reason for the differ-ences in colonization of skin ulcers of lep-rosy and control patients could be the selec-

64, 3^Sturm, et al.: Microbial Flora of Leprosy Ulcers^279

tion of organisms resistant to the drugs usedfor the treatment of leprosy. Therefore, dap-sone (sulfonamide) and rifampin resistancewas examined. All branching rods andgroup G streptococci were resistant to sul-fonamides hut so were all of the other or-ganisms in the leprosy as well as the controlgroup. Resistance to rifampin showed amore complicated picture. There was a sig-nificantly higher level of resistance amongdifferent groups of bacterial isolates fromleprosy patients, including group G strepto-cocci. This difference is, however, based ona broad range of MIC values and not onhigher MICs for all isolates. The branchingrods were fully susceptible as were 15 outof the 19 group G streptococci; the other 4showed high-level resistance. These fourwere responsible for the high mean MICand highMIC )) values. Susceptibility testsfor clofazimine were not performed. Al-though resistance to the antileprosy drugswas found with higher frequency in the lep-rosy group, it does not provide an explana-tion for the observed difference in coloniz-ing species. The presence of drug-suscepti-ble strains (branching rods) cannot havebeen due to the selective pressure of the an-tibiotics used.

Leprosy ulcers showed a low mean in-flammatory score (IS) as compared to thecontrol group, the IS of the latter being inaccordance with the findings of others ( 7 ).The presence of pyogenic streptococci to-gether with S. aureus resulted in a lower ISthan either of these separately, while thepresence of A. haemolyticum or unidenti-fied branching rods together with faculta-tive gram-negative bacilli enhanced inflam-mation. The mechanism of these bacterialinteractions is unclear and warrants furtherstudy. If a high IS is related to the develop-ment of cellulitis and osteitis of adjacenttissue, leading to systemic disease and de-formity, regular IS evaluation with timelyintervention could prevent this. Based onobservations of this study, the aim shouldhe continuous ulcer care to reduce the num-ber of colonizing bacteria and antimicrobialtreatment when there is a rise in the inflam-matory reaction. The number of colonizingorganisms also can be kept low by applyingdisinfecting dressings, while the choice ofantimicrobial drugs should focus on entero-bacteria as well as pyogenic cocci. A com-

bination of a fluorinated quinolone withpenicillin would be adequate. However, theefficacy of such an approach has yet to beestablished.

SUMMARYThe microflora of 55 patients with lep-

rosy skin ulcers was studied and related to aweighted inflammatory score (IS). The con-trol group consisted of 18 ulcers with dif-ferent underlying pathology. Leprosy ulcerswere characterized by the exclusive pres-ence of two types of branching gram-posi-tive rods; a particular interesting proposal isthat Mycobacterium leprae share commonantigens with these unusual "leprosy ulcerassociated" organisms and group G beta-hemolytic streptococci. In the leprosygroup, corynebacteria and branching rodsaccounted for 97% of gram-positive bacilliand Bacillus species constituted only 3%.In the control group, B. species formed 50%of gram-positive rods; the rest werecorynebacteria (p = 0.03). In the leprosygroup, one third of the gram-positive bacte-ria were branching rods; none of them wasacid fast. Ten of them were identified as Ar-canobacterium haemolyticum, and the re-maining 7 could not be identified. The IS ofleprosy patients was lower than in the con-trol group. The presence of more than twospecies of facultative or aerobic gram-neg-ative rods or single species of pyogenicgram-positive cocci correlated with a highIS. The presence of two or more differentpyogenic cocci resulted in a lower IS. Fur-ther studies into the nature of leprosy-unique organisms as well as the inflamma-tion inhibition factors in mixed infectionsare warranted. It is recommended that man-agement of ulcers should consist of the ap-plication of local disinfection and earlytreatment of episodes of inflammation witha combination of fluoroquinolone and peni-cillin.

RESUMENSe estudió la microOora de las Meeras de piel de 55

pacientes con lepra y se relacion6 con el registro delpeso inflamatorio (1)1). Li grupo control cstuvo confor-mad() por I8 pacientes con tileeras de patologias diver-sas no leprosas. Las Mceras de la lepra se caraeteri-zaron por la exclusiva presencia dc 2 tipos de baci losramiticantes Gram-positivos. Se propone que My-

280^ hitemational Journal of Leprosy^ 1996

co/mete/1/mi /eprae comparte antigenos commies conestos organismos asociados con las dIceras de la lepray con estroptococos beta-hemoliticos del grupo G. Enel grupo de lepra las corinebacterias y los bacilos ram-incomes constituyeron el 97% de los bacilos Gram-positi vos y las especies del genero Bacillus sólo el 3%.En el grupo control, las especics de Bacillus fOrmaronel 50% de los bacilos Gram-positivos, el resto fueroncorinebacterias (p = 0.03). l'.11 el grupo de lepra, tm ter-cio de las hacterias Gram-positivas fueron bacteriasramificantes; ninguna de ellas fue acido-resistente.Diez de ellas fueron identificadas como Arcanobac-terium haemolyticum y los 7 restantes no pudieronidentificarse. El PI en los pacientes con lepra Inc Indsbajo que el PI en el grupo control. La presencia de Indsde 2 especies de hacilos facultativios o acrobicosGrain-ncgativos o do especies tinicas de corns pió-genus Gram-positivos correlaciona con un P1 elevado.Se requieren nuis estudios sobre la naturaleza de losorganismos asociados a la lepra y sobre los factores in-hibitorios de inflamación en las infecciones mixtas. Serecomienda que el manejo de las Ulceras incluya Iadesinfección local y el tratamiento temprano de losepisodios de inflamación con una combinación de fiu-oroquinolona y penicilina.

RÉSUMÉ

On a étudie la microflorc de 55 patients avec des ul-ceres cutanes lépreux, en relation avec un score in-flammatoire (SI) pondéré. Le groupe temoin se coin-posait de 18 ulceres avec inverses maladies sous-ja-centes. Les ulcéres lépreux se caractérisaient par hipresence exclusive de deux types de baguettes nutle-t -lees gram-positives; une proposition particuliérementinteressante est que Mycobacterium leprae portage desantigénes communs avec ces organismes inhahituels"associes aux ulcéres lépreux" et des streptocoquesbeta hémolytiques du groupe G. Dans le groupe lepre,les corynebacteries et les baguettes ramétiées comp-taient pour 97% des hacilles gram positifs et Bacillusspecies seulement pour 3%. Dans le groupe temoin, B.species formait 50% des baguettes grain positives; lereste emit des coryne bacteries (p = 0.03). Dans legroupe lepre, un-tiers des bactéries grain positives emitdes gahuettes raménées; aucune d'entre elles ne rCsis-tait a l'acide. Dix &entre elles ont été identitiées entant que Anyozobacteriton haemolvticutm et les 7autres Wont pu étre identifiées. Le SI des patientslépreux était plus foible que cent' du groupe temoin.La presence de plus de deux especes de baguettesgram negatives facultatives ou aérobiques on dunesettle espéce de coques gram positifis pyogénes emitassocie 3 un SI élevé. La presence de deux coques pyo-geniques differents ou plus resultait dans tin SI plusfoible. Des etudes ulterieures se rapportant a la naturede ces organismes uniques it Ia lepre ainsi que stir lesfacteurs d'inhibition de [inflammation dans les infec-tions mixtes vont titre féalisées. On recommande que

le traitement des ulceres CoIlsiste en l'applicationdune desinfection locale ainsi que le traitment precocedes episodes d' [illumination par une combinaison defluoroquinolone et de penicilline.

Acknowledgment. 'this study was financiallysupported by LEPRA, the British Leprosy Relief As-sociation, through a grant to AWS.

REFERENCESI. ANTIA, N. H. 'Fite people we fail to reach. Lepr.

Rev. 48 (1977) 155-156.2. ArsroNIE, 1., Coi:NE, M. and Cocrro, C. Site and

homology of the genomes of leprosy-derivedcorynebacteria, Mycobacterium leprae and othercorynebacteria and mycohacteria. J. Med. Micro-biol. 27 (1988) 45-50.

3. BEctitArr, L. M. and MARTINEZ DOMINQUEZ, V.The leprosy problem in the world. Bull. WHO 34(1966) 811-826.

4. BROWN, S., LANELI.E, M. A., ASSELINEAU, J. andBARKsonLE, L. Description of Cmynebacteriion(I/ben:Was/Tar/cum sp. nov., a leprosy-derived co-rynebacterium. Ann. Microbiol (Institut Pasteur)1358 (1984) 251-267.

5. ClIAKRABARTY, A. N., DAsTioAR, S. G., DAS, S.and CitAUDIBIRY, S. K. Repeated isolation of No-canlia-like organisms from multibacillary leprosy.Indian J. Lepr. 59 (1984) 247-262.

6. Cocrro, C. and DELvirii, J. Properties of mi-croorganisms from human leprosy lesions. Rev.Infect. Dis. 5 (1983) 649-657.

7. EIIRENKRANZ, N. J., AIrroNso, B. and NEZENBERG,D. Irrigation-aspiration for culturing draining de-cubitus ulcers: correlation of bacteriological find-ings with a clinical inflammatory scoring index. J.Clin. Microbiol. 28 (1990) 2389-2393.

8. GUEUR, M. C., HARBOE, M., FONTAIN, F., DEL-VILLE, J. and Como, C. Comparison of the cyto-plasmic antigens of leprosy-derived corynebac-teria and some mycohacteria. Scand. J. Immunol.17 (1983) 497-506.

9. lloosEN, A. A., RAs(x)i., M. N. and Roux, L. Posttraumatic ankle joint infection with Arcanobac-terium haemolvticum: a case report. J. Infect. Dis.162 (1990) 780-781.

10. ISENBERG, H. D. Clinical Microbiology Proce-dures Handbook. Washington, D.C.: American So-ciety for Microbiology, 1992.

I I. JANCZURA, E., Anou-ZEID, C., GAII.I.Y, C. andCocrro, C. Chemical identification of sonic cellwall components of microorganisms isolated fromhuman leprosy lesions. Zentralbl. I3akteriol. 251(1981) 114-125.

12. KRIEG, N. R. and Hour, J. G. Bergey's Manual ofSystemic Bacteriology Vol. I. Baltimore: Williamsand Wilkins, 1984.

64, 3^Sturm, et al.: Microbial Flora of Leprosy Ulcers^281

13. LAUB, R.,^J. and Coca°, C. Immuno-logical relatedness of ribosomes from mycobacte-ria, nocardial and corynebacteria, and microorgan-isms in leprosy lesions. Infect. Immun. 22 (1978)540-547.

14. Pullin's, I. A guide to sensitivity testing. J. An-timicrob. Chemother. 27 Suppl. D (1991) 1-48.

15. RIDLEY, D. S. Histological classification and theimmunological spectrum of leprosy. 13u11. WHO51 (1974) 451-464.

16. SNEATII, R II. A., MAIN, N. S., SHARPE, M. E. andHort., J. G. Bergey:c Manual of Systemic Bacteri-ology Vol. 2. Baltimore: Williams and Wilkins,1986.

Page 1Page 2Page 3Page 4Page 5Page 6Page 7Page 8