As result of the studies undertaken during the last 4 years at the Tuberculosis

Chemotherapy Centre, Madras, some characteristics of Indian tubercle

bacilli which differ from those of British tubercle bacilli have been observed. These

factors relate to their virulence in the guinea-pig, susceptibility to hydrogen peroxide,

and drug sensitivity. The Indian and British cultures are, however, closely

similar in the majority of their in vitro characteristics.

Before proceeding further, it must be emphasized, that the findings reported

in this paper apply to cultures obtained from a rather selected group of patients.

These patients presented themselves because of symptoms and the vast majority

of them were positive on smear examination and had moderately or far advanced

disease. Furthermore, all the patients were residents of Madras city

Bhatia, A L

NIRT Institutional Repository

Ind. Jour. Chest Dis., III 3, 1961, pp. 147-153.CHARACTERISTICS OF INDIAN TUBERCLE BACILLI*BYA.L .BHATIA†As result of the studies undertaken during the last 4 y ars at the Tuber-culosis Chemotherapy Centre, Madras, some characteristics of Indian tuberclebacilli which differ from those of British tubercle bacilli have been observed. Thesefactors relate to their virulence in the guinea-pig, susceptibility to hydrogen per-oxide, and drug sensitivity.The Indian and British cultures are, however, closelysimilar in the majority of their in vitro characteristics.Before proceeding further, it must be emphasized, that the findings reportedin this paper apply to cultures obtained from a rather selected group of patients.These patients presented themselves because of symptoms and the vast majorityof them were positive on smear examination and had moderately or far advanceddisease. Furthermore, all the patients were residents of Madras city.Virulence:It had previously been suggested by Dhayagude and Shah (1948), Frimodt-Moller et al (1956), and Singh (1967), that a proportion of Indian cultures oftubercle bacilli were less virulent for guinea-pigs than European strains. It waspossible, however, that this difference in the guinea-pig virulence of Indian tuberclebacilli could have been due to one of the following :1. The difference in the breed of animal used in India as compared toEurope.2. The environmental and dietary conditions under which the animals arekept in India.3. The difference in the technique used, or4. that the strains tested were not typical tubercle bacilli, but anonymousmycobacteria.The problem of virulence in the guinea-pig of Indian tubercle bacilli was,therefore, re-investigated, with specific emphasis on the elucidation of the roleof these possible factors. The experiments were so designed that cultures fromIndian patients in Madras and British patients in England tested in 3 labora-tories-2 in England and 1 in Madras in 3 breeds of guinea-pigs-1 bred inIndia for a long time at Madanapalle, 1 brought out from England and used bothin London and Madras, and the 3rd bred at Porton, Salisbury, England. The2 Some Characteristics of lndian Tubercle Bacillicultures were flown from India to England and vice versa.All the cultures weretested by a series of in vitro identification tests to ensure that they were typicalmammalian tubercle bacilli.The virulence in the guinea-pig were determined from the extent of the diseasepresent at 6 and 12 weeks after inoculating 1 mg. moist weight of bacilli intramuscu-larly. The extent of the disease was assessed by a scoring procedure, the possiblescores ranging from 0 to 100.The best measurement of virulence was found to be an index obtained bydividing the score by the survival time of the animals in days (Mitchison et al, 1960).It indicated the rate of development of lesions and also by inference the rate ofmultiplication of the bacilli in the organs of the guinea-pig. It also allowed compari-son of the results, irrespective of whether death had occurred before the dayappointed for autopsy.No significant difference was found between the different experiments and therewas no marked difference between the different breeds of the guinea-pigs or betweenthe assessment carried out in different laboratories.In addition to the comparisonof the post-mortem scores and the indices of virulence, 2 other methods wereemployed to compare the virulence.One was the mortality of guinea-pigs and theother was the frequency and degree of positivity of cultres from the spleen ofguinea-pigs infected with Indian cultures, as compared with those infected withBritish cultures. All these 3 methods showed that Indian cultures differ fromBritish cultures in that they have a wider range of virulence, approximately 30 percent being as virulent as the British cultures, 40 per cent having an intermediatedegree of virulence and 30 per cent being markedly attenuated.The frequency distribution of the mean indices of virulence of the 49 Indianand 10 British pretreatment drug-sensitive cultures studied by Mitchison et al(loc. cit.) is presented in Table I.The mean index of virulence is the averageof the 6-week and 12-week indices. While none, of the 10 British cultures had amean index of virulence of less than 0.8, 34 of the 49 Indian cultures were in thisgroup. Conversely, all 10 British cultures had a mean index of more than 0.8 ascompared to only 15 of the 49 Indian cultures.The mean index of virulence forthe Indian cultures was 0.64, compared with 1.12 for the British cultures. Thisdifference between the Indian and British cultures attains statistical significance(P<0.001).The practical implications of this finding are :-First, in India reliance cannot be placed on the guinea-pig as a means ofisolating tubercle bacilli diagnostically; and in particular from specimens in whichthe bacilli are expected to be present only in small numbers.Secondly, the identification of Indian cultures of tubercle bacilli as Mycobac-terium tuberculosis cannot be based on their virulence for the guinea-pig.A. L. Bhatia 3TABLE IVirulence in the guinea-pig of pretreatment Indian and Britishcultures of tubercle bacilli10 (100)1.12*Brackets indicate percentages based on fewer than 25 observationsThere is one rather interesting possible explanation of this finding of so manyattenuated strains in India.S nce there is no evidence that the Indian strains areless infective than the British strains, it is possible that these attenuated culturesexist in India, because under the conditions applying here, they have a relativebiological advantage over virulent strains. In this population, with high risk ofinfection and with malnutrition, the attenuated strains may more often be able tocause chronic cavitating disease and thus continue to exist. Attenuation may,therefore, be a mechanism to establish a balance between the susceptibility of thehost and the virulence of the parasite.It had also been shown (Bhatia et al, 1961) that cultures obtained from Indianpatients at different times have a consistent degree of virulence. Thus, variation invirulence from culture to culture obtained at intervals of upto 6, weeks from thesame untreated Indian patient, was no greater than the natural variation in responseof the guipea-pigs in the virulence test and was considerably less than the variationin virulence from patient to patient.It was, therefore, meaningful to correlatedifference in virulence of tubercle bacilli obtained from Indian patients before thestart of chemotherapy with the severity or type of the disease and their subsequentresponse to treatment with anti-tuberculous drugs. In brief, there were no impor-tant associations between pretreatment virulence and the severity of the disease onadmission to treatment, as assessed by total extent of the radiographic lesions, theextent of cavitation and the degree of positivity of the sputum (Tuberculosis Chemo-therapy Centre, 1960).Further findings of the relationship between the virulenceof pretreatment cultures and response to chemotherapy will be presented later.Susceptibility to hydrogen peroxide :Indian and British cultures of tubercle bacilli have also been compared inrespect of their susceptibility to hydrogen peroxide (Subbaiah et al 1960). The4 Some Characteristics of Indian Tubercle Bacillimethod briefly was to expose cultures to 0.02 per cent hydrogen peroxide for 90minutes and then to determine by viable counting the number of bacilli thatsurvived. The number of organisms which survived exposure to hydrogen peroxidehas been expressed as a percentage of the total number of organisms that wereexposed.The susceptibility to hydrogen peroxide of 8 British and 7 Indian cultureswhich survived exposure to hydrogen peroxide for 90 minutes is shown in Table II.All the cultures had 2 plus, that is full catalase activity and all were sensitive toisoniazid. All the 8 British cultures were relatively resistant to hydrogen peroxide,32 per cent or more of the organisms being viable after exposure for 90 minutes.Of the 7 Indian cultures tested; 4 were markedly susceptible, 2 per cent or less ofthe organisms surviving.T ABLE  I ISusceptibility hydrogen peroxide of Indian and British cultures of tubercle bacilliCulture No.Percentage oforganisms resistantto hydrogen peroxideIsoniazid-resistant organisms with reduced catalase activity are known to beattenuated in the guinea-pig. These organisms are also more susceptible toexposure to hydrogen peroxide.It is, therefore, possible that the difference in thevirulence in the guinea-pig of different Indian cultures of tubercle bacilli resultsfrom the differences in their susceptibility to hydrogen peroxide.This problem inat present being investigated.Drug sensitivity:The Indian strains differ from British strains in their sensitivity to PAS and thiacetazone.Sensitivity tests to PAS were performed by the method described in detail inan earlier report from this Centre (Tuberculosis Chemotherapy Centre, 1959).A. L. Bhatia 5In brief, a representative sample of the cultures was inoculated on to a series ofincreasing concentrations of PAS in Lowenstein-Jensen medium; After 4 weeksincubation at 37°C, the degree of growth was examined. The lowest concentrationof PAS which inhibited the growth of the culture was determined using 2 differentdefinitions of growth on the drug containing slopes.(1) Significant growth was defined as 20 or more colonies ; this is referredto as the 20-colony minimal inhibitory concentration (MIC).(2) Significant growth was defined as presence of 100 or more colonies ;this is referred to as the 100 colony MIC.Table III presents a comparison of the sensitivity to PAS of pretreatment Indianand British cultures of tubercle bacilli. Considering 20-colony MIC values, theIndian strains were on the average more resistant than the British strains.Valueof 4 µg/ml or more were given by 38 (47 per cent) of the 81 Indian strains and byone (2 per cent) of the 52 British strains.In addition, the range of the MIC valuesfor Indian cultures was wider than that for British cultures. The difference betweenIndian and British cultures was markedly reduced, however, when the 100 coloniesdefinition of growth was used. Minimal inhibitory concentrations of 2µg/ml or morebeing required by 11 (14 per cent) of the 81 Indian strains and by one (2 per cent)of the 52 British strains.TABLE IIIFurther investigations on the PAS sensitivity of tubercle bacilli (Selkon et al,1960) have confirmed that this difference between the PAS sensitivity of Indianand British cultures is genuine.It appears that thin difference is due to the fact thatIndian cultures of tubercle bacilli contain a small proportion (0.02 per cent orslightly less) of PAS resistant organisms which are not present in British cultures.6 Some Characteristics of Indian Tubercle BacilliThis in vitro difference between British and Indian cultures does not appear,however, to be of any importance in viva We have not been able to observe anyrelationship between the presence of this small proportion of resistant strains andany tendency for the patient to fail to respond to treatment with PAS.The exacttype of in vitro sensitivity test against PAS for Indian cultures has not as yet beendetermined, but it would seem that the use of a smaller inoculum, namely, 1: 10 ofthe normal inoculum may be suitable.An even more marked difference exists between Indian and British cultures,in their behaviour in the thiacetazone sensitivity tests (Thomas et al, 1961). Indiancultures showed considerable variation in their sensitivity to thiacetazone whichplus partly due technical variation, but also in part due to differences in theirsensitivity to this drug.The thiacetazone sensitivity test, therefore, cannot be usedas an identification test for tubercle bacilli, as in the case in Europe.In viva characteristics :Having mentioned the differences between Indian and British cultures, it isimportant to emphasise their similarity and the important fact that cultures obtain-ed from patients’ resident in Madras city with clinically diagnosed pulmonarytuberculosis and invariably infected with typical human tubercle bacilli.We admitted 341 patients to a controlled study of 4 regimens of domiciliarychemotherapy (Tuberculosis Chemotherapy Centre, 1980). Cultures from 287 ofthese 341 patients were examined by series of in vitro identification tests.These  t e s t s  w e r e : - 1 .  B a c t e r i a l  m o r p h o l o g y -2. Colonial morphology on(a) Lowenstein-Jensen slopes(b) 7H-10 oleic acid albumen agar plates3.. Growth at 23°C.4. Pigmentation after exposure to light6. Catalase activity of growth on (a) a drug-free Lowenstein-Jensen slope(b) an a slope of Lowenstein-Jensen medium containing 50 µg/mli s o n i a z i d6 .  N i a c i n  p r o d u c t i o n ,Cultures from the remaining 64 patients were tested for their sensitivity to.isoniazid, catalase activity and photochrmogenicity.In addition, a few cultureswere also, tested for guine-pig virulence.The investigation has been described indetail by Thomas et al (loc. cit.). All the 341 patients admitted to this trial yieldedcultures which on the basis of these investigations were regarded as human tuberclebacilli. Of the 341 cultures examined, 2 were anonymous mycobacteria, but neitherof them were regarded as the aetiological agent of the patients’ disease.I n  b o t hcases,these anonymous mycobacteria were found on only 1 occasion from these;A. L. Bhatia 7patients and other cultures were obtained from them, which were typical tuberclebacilli. The absence in this series of cases, of any instances in which anonymousmycobacteria could be incriminated, as the aetiological agent of clinically diagnosedpulmonary tuberculosis is of particular interest because of the relative frequency withwhich they are encountered in other countries particularly some parts of the U.S.A.Niacin production was tested for in 277 of the 341 Indian cultures. Allcultures were niacin positive and have, therefore, been regarded as human strains.This is a little surprising in view of the fact that 1.8 per cent of the cattle and2.8 per cent of the buffaloes in Madras City, and adjacent rural areas have beenshown to be tuberculin reactors.Conclusion :In conclusion, studies on Indian-cultures of tubercle bacilli have shown :-1. They have a wider range of virulence in the guinea-pig than Britishcultures, approximately 30 per cent being as virulent as British cultures.2. A proportion of Indian cultures show greater susceptibility to hydrogenperoxide than the British cultures.3. Indian cultures behave differently to British cultures in the standardsensitivity tests to PAS and thiacetazone.4. The aetiological agent of clinically diagnosed pulmonary tuberculosis inpatients resident in Madras city is Mycobacterium tuberculosis var. hominir.Acknowledgement :My thanks are due to the Indian Council of Medical Research for permittingme to present this paper.REFERENCESDHAYAGUDE , LG., and SHAH, B.R. (1948)Fwatoor-MOLLER, J., MATHEW. K.T. (1956)and BARTON, R.M.MITCHISON , D.A., WALLACE . J.G. (1960)BHATIA , A.L,. SELTON , J.B.SUBBIAH, T.V. and LANCASTER ,M.C.S E L K O N, J.B.. SU B B I A H , T.V., (1960)BHATIA , A.L.. RADHAKRISHNA ,S. and MITCHISON , D.A.SINGH. B. ( 1957)T.V., MITCHISON, D.A. (1960)OSEPH S., SUBBAIAH, (1961). 9Tubercle (Lond.). 42, 317.Ind J. Med. Res. 36, 79.Proceedings of the Thirteenth TuberculosisWorkers’ Conference. p. 157. Tubercu-losis Association of India, New Delhi.Tubercle (Lond.). 41, 1.Bull. Wld. Hlth Org. 23, 599.

Charateristics of Indian Tubercle Bacilli

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Charateristics of Indian Tubercle Bacilli

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