35~ Tubercle, Lond., (1964), 45, 354

V I R U L E N C E IN T H E G U I N E A - P I G A N D S E N S I T I V I T Y T O P A S A N D

T H I A C E T A Z O N E O F T U B E R C L E B A C I L L I F R O M S O U T H I N D I A N

P A T I E N T S W I T H P U L M O N A R Y T U B E R C U L O S I S

By S. JOSEPH, D. A. MITCHISON,* K. RAMACHANDRAN, J. B. SELKON+ and T. V. SUtmAIAH~

fi'om Tttberctdosis C/tentotherapj, Centre..~ Madras. htdia

SUMMARY

lsoniazid-sensitive strains of tubercle bacilli, obtained pretreatment from 280 South Indian patients, were tested for their virulence in the guinea-pig and for their sensitivity to PAS; strains from 209 of these patients were also tested for their sensitivity to thiacetazone.

Strains of low virulence more often had a high proportion of PAS-resislant mutants and were more often resistant to thiacetazone than those of high virulence.

There is suggestive evidence that Indian strains can be divided into two groups, the smaller group, comprising about one-quarter of the strains, resembling strains from British patients and the larger group characterized by a lower virulence, a higher susceptibility to llydrogen peroxide, a higher proportion of PAS-resistant mutants and thiacetazone resistance.

Isoniazid-sensitive cultures of tubercle bacilli obtained from South Indian patients with pulmonary tuberculosis before the start of antituberculos~s chemotherapy have been sllown to differ from corresponding cultures from British patients in 4 respects: (I) The Indian cultures were less virulent, on average, and had a wider range of virulence in the guinea-pig than British cultures (Frimodt-Moller, Mathew and Barton, 1956; Frimodt-Moller, 1957: Mitchison and others, 1960; Bhatia and others, 1961); (2) The Indian cultures were more susceptible to the bactericidal activity of low concentrations of hydrogen peroxide (Subbaiah, Mitchison and Selkon, 1960); (.3) The Indian cultures contained a higher proportion of mutants resistant to p-aminosalicyclic acid (PAS) (Selkon and others, 1960); (4) The Indian cultures were more resistant to thiacetazone (Tb.omas and others, 1961; Mitchison and Lloyd, 1964). An association has been found in Indian cultures between virulence in the guinea-pig and peroxide susceptibility (Mitchison, Selkon and Lloyd, 1963; Nair and otllers, 1964). It is therefore of interest to see whether associations exist in Indian cultures between virulence and sensitivity to PAS and thiacetazone.

Methods Strains o f Tubercle Bacilli

Virulence tests in the guinea-pig were done on a single culture obtained from the sputum of each of 280 Sou'th Indian patients with pulmonary tuberculosis on admission to a chemotherapy study (Tuberculosis Chemotherapy Study, Madras, 1960) The isolation of the cultures and the

*Present address: Postgraduate Medical School of London, Ducane Road, London, W.12. tPresent address: Public Health Laboratory, Newcastle-upon-Tyne. ++Present address: Department of Biochemistry, Albert Einstein College of Medicine, Bronx 61. New York. ~The Centre is under the joint auspices of the Indian Council of Medical Research, the Madras State Government,

the World Health Organization and the Medical Research Council of G'reat Britain.

SOUTH INDIAN TUBERCLE BACILLI 355

arrangement of the virulence tests have been fully described by Mitchison and others (1961). The patients had not received more than two weeks of previous chemotherapy (the great majority had none) and their cultures were sensitive to isoniazid. PAS sensitivity tests were done on a pretreat- ment culture from all of the 280 patients; in 236 patients the same culture was tested as was used for the virulence test, and in 44 patients separate cultures, obtained within a few days of each other, were used for the two tests. Of the 280 cultures with virulence tests, 209 were also tested for their sensitivity to thiacetazone. The results of the thiacetazone sensitivity tests are included in those reported by Thomas and others (1961).

Sputum specimens were obtained from newly diagnosed, previously untreated British patients with pulmonary tuberculosis attending selected Chest Clinics in Great Britain. The results of PAS sensitivity tests on cultures from 52 of these patients have been described previously (htvestigation ! of Selkon and others, 1960), as have the results of the thiacetazone sensitivity tests on cultures from 66 patients (Thomas and others, 1961). These results are included for comparison with the results on Indian cultures in the present report.

Virulence Tests Virulence tests were carried out at the Microbiological Research Establishment, Porton (Porton)

and at the Tuberculosis Chemotherapy Centre (Madras). The measure of virulence used was based on the rate of progression of the disease in the guinea-pig and has been fully described by Mitchison and others (1960, 1961). In brief, 1 mg. (moist weight) of the culture was inoculated intramuscularly into each of two or four guinea-pigs, half of which were killed at 6 weeks and the other half at 12 weeks. At the post-mortem examination, the total extent of tuberculosis disease was assessed as a score ranging from 0 to I00. The squa."e-root of the ratio of the score to the survival period in days was determined for each guinea-pig (whether sacrificed or dead from tuberculosis) and was termed the 'root-index'. The root-index of virulence was defined as the mean of the root-indices for the two or four guinea-pigs inoculated with each culture, and has been used as the measure of virulence of the culture. The responses to infection were different in the breed of guinea-pig used at Porton and in the breed used at Madras. The results of the smaller Madras series were adjusted to those in the Porton series by the method described by Mitchison and others (1961).

Sensitivity Tests In the sensitivity tests, a suspension was prepared by adding approximately 2 mg. (moist weight)

of a representative sample of the bacillary mass from a culture on L/Swenstein-Jensen medium (the medium did not contain potato starch, Jensen, 1955) to 0.5 ml. sterile water in a 7 ml. (�88 oz.) screw-capped bottle containing about six beads of 3 ram. diameter, and then shaking the bottle mechanically for one minu1-" A 3 mm. loopful of this suspension was inoculated on to slopes of L6wenstein-Jensen medium , .,1raining the following drug concentrations:

0, 2, 4, 8, 16 and 64 !J: ' ~dium PAS dihydrate. 0, 0.25, 0.5, 1, 2, 4, 8. ~, ano 12 ,u,g/ml, thiacetazone. The slopes used in ~l~e thi:,, tazone tests all contained I ~ triethylene glycol, necessary

for solution of this drug. PAS anti thiacetazone tests were set up on different occasions from separate suspensions. The tests were incubated at 37~ and read at 4 weeks. 'Growth ' on a slope was defined as (I) the presence of 20 or more colonies or (2) the presence of 100 or more colonies. The lowest concentration of the drug inhibiting each of these degrees of growth was recorded ' as an approprihte minimal inhibitory concentration (MIC). Thus two end-points were obtained for each test, which will be referred to as the 20-colony MIC and the 100-colony MIC.

Results Virulence and Sensitivity to PAS

The root-indices of virulence and the sensitivity to PAS of pretreatment strains from 280 Indian patients are set out in Table I together with the results of PAS sensitivity tests on cultures from 52

356 TUBERCLE

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SOUTH INDIAN TUBERCLE BACILLI 357

British patients. The PAS sensitivity tests on the British cultures were done at the Centre by the same method as was used for the Indian cultures, but at a later date.

Cultures with root-indices of virulence of 0-20-0.59 were of low virulence, usually producing naked-eye lesions in the guinea-pig only at the site of inoculation or in its draining lymph glands. Cultures with root-indices of virulence of 0.80 or more caused extensive, progressive disease.

Although the 52 British cultures were not tested for their virulence, a further sample of 65 British cultures, obtained under the same circumstances and tested in comparison with the 280 Indian cultures used in the present study, v~ere all found by Bhatia and others (1961) to have root-indices of virulence of 0"80 or more; the mean root-index was 1.05.

A 20-colony MIC of 4 ~g/ml. sodium PAS or more was found in 59~ of 180 Indian strains with root-indices of virulence of 0.20-0.79, in 36 ~ of 58 strains with root-indices of virulence of 0.80-0.99, and in 17 ~ of 42 strains with root-indices of virulence of 1.00 or more. The association in the Indian strains between virulence and sensitivity to PAS is significant (Z2(.~)--29-18, P < 0.001). Only 2 ,~ of tile 52 British cultures had a 20-colony MIC of 4 ~g/ml. or more, a proportion similar to that found in the most virulent of the Indian strains.

The 100-colony MIC of sodium PAS was 4 ~tg/ml. or more in 12~ of 180 Indian strains with root-indices of virulence of less than 1.00 and in 4 ~ of 100 Indian strains with root-indices of virulence of 1.00 or more, a difference that does not attain statistical significance. Although definite conclusions cannot be drawn because slopes containing less than 2 ~.g/ml. sodium PAS were not included in the sensitivity tests, there is no clear evidence of an association between virulence and sensitivity to PAS using the 100-colony end-point. None of the 52 British cultures had an MIC of 4 ~g/ml. or more.

I/irulence and Sensitivity to Thiacetazone The root-indices of virulence and the sensitivity of the pretreatment cultures from 209 Indian

patients are set out in Table II with the results of thiacetazone sensitivity tests on 66 cultures from British patients. The thiacetazone sensitivity tests were done at about the same time on the Indian and the British cultures.

A 20-colony MIC of 2 ~tg/ml. thiacetazone or more was obtained in 7 6 ~ of 131 Indian cultures with root-indices of virulence of 0.20-0.79, in 61 ~o of 41 cultures with root-indices of virulence of 0-80-0.99, and in 30 ~ of 37 cultures with root-indices of virulence of 1.00 or more. The association between virulence and thiacetazone sensitivity in the Indian cultures is significant (Z~(2)--27.95, P < 0.001). The 20-colony MIC was 2 ~.g/ml. or more in 17~o ofthe 66 British cultures, a proportion similar to that found with the most virulent of the Indian cultures.

Aft association of similar degree is evident between the virulence of the Indian cultures and their sensitivity to t h i a ~ z o n e in the 100-colony readings. Again, the sensitivity of the British cultures corresponds closely to the sensitivity of the more highly virulent Indian cultures.

Sensitivity to Thiacetazone and to PAS Estimates of the 20-colony MICs of thiacetazone and of PAS were available for 205 of the

strains from Indian patients (Table I!I). An MIC of 4 ~g/ml. sodium PAS or more was obtained in 26 (36~) of 73 strains with an MIC of less than 2 ~tg/ml. thiacetazone and in 71 (54~) of 132 strains with an MIC of 2 ~.g/ml. thiacetazone or more" the difference issignificant (P---0-02). Considering higher degrees of PAS-resistance, an MIC of 16 ~tg/ml. sodium PAS was found in none of the 73 strains more sensitive to thiacetazone and in 25 (19~o) of the 132 strains more resistant to thiacetazone.

Discussion

Selkon and others (1960) compared the sensitivity to PAS of cultures of tub erc-le bacilli from South Indian and British patients by the same methods that were used in the present"s'tudy. High 20-colony MICs were more often found with the Indian cultures than with the British cultures,

358 TUBERCLE

TABLE [[|.~SENS1TIVITY TO PAS Ant)TO THIACETAZONE OF STRAINS FROM INDIAN PATIENTS (20-COLONY END-POINIXS)

M I C of l]liacela2olle

(~zg/ml.)

0.5 or les:~ 1

2 4

8 or more

Total

Total strains

38 35 90

' 34 8

2 , or less

29 5 18 12 42 15 17 3 2 2

205 108

M I C o f sodium PA S ('~J.g/ml.)

8 16

4 0 5 0

15 !1 8 4 3 0

35 15

32 or more

4 or i~lOl'e No. %

0 9 24 0 17 49 7 48 53 2 17 50 1 6 (75)*

10 97 47

*The percentage in parenthesis is based on fewer than 25 observations.

but the difference in the 100-colony MICs was small or not evident. They concluded that the Indian cultures contained a higher proportion of PAS-resistant mutants than the British cultures and that these resistant bacilli in the Indian cultures yielded 20 or more colonies on slopes containing the higher concentrations of PAS when the inoculum in the sensitivity tests was, by chance, large. However, even with a large inoculum, they rarely yielded as many as 100 colonies. In the present study strains of low virulence in the guinea-pig more often had fiigh 20-colony M ICs than strains of high virulence, but from the limited evidence available, there did not appear to be an appreciable difference between them in the values of the 100-colony MIC. Thus it appears likely that it is the Indian strains of low virulence that have a high p r o p o r t i o n o f resistant mutants, whereas sirains of high virulence contain a lower proportion of mutants and are similar to British cultures in their pattern of sensitivity to PAS.

The Indian strains of low virulence were also more often resistant to thiacetazone than the strains of high virulence. However, the association between virulence and thiacetazone sensitivity was similar in degree with both. the 20-cdlony MIC and the 100-colony MIC values. Thus resistance to thiacetazone appears to be a property of a larger proportion of the bacterial population in the strains of low virulence than is resistance to PAS. The thiacetazone sensitivity of the Indian strains of high virulence and of the British strains was similar. An association between virulence and thiacetazone sensitivity in a smaller number of Indian strains has also been found by Mitctaison and Lloyd (1964).

Mitchison, Seikon and Lloyd (1963) and Nair and others (1964) have found an association between the virulence in the guinea-pig of strains from Indian patients and the proportion of the organisms that survive exposure to hydrogen peroxide. There is also a suggestion from the data of the latter authors that the strains can be divided, principally because of bimodality in the distribu- tion of peroxide Susceptibility, into two groups, the larger, comprising nearly three-quarters of the strains, with low virulence and high susceptibility to peroxide and. the smaller with high virulence and low peroxide susceptibility. The findings of the present report suggest that the strains in the larger group not only have low'virulence and high peroxide susceptibility, but also tend to be resistant to thiacetazone and to have a high proportion of PAS-resistant m u t a n t . The smaller group of strains resemble cultures from Britain in virulence, peroxide susceptibility and sensitivity to thiacetazone and PAS. The failure to distinguish clearly between these two groups (if they are really separable) in the data relating virulence to thiacetazone sensitivity may be due to the low precision of the virulence, test, to the small difference in mean thiacetazone sensitivity between the groups and to the considerable variation inherent in the method of testing thiacetazone sensitivity, exemplified in the results of tests done on duplicate pretreatment cultures from East

SOUTH INDIAN TUBERCLE BACILLI 359

African patients (East African/Brit ish Medical Research Council, 1960, 1963). Fur thermore , even if all of the Indian strains of low virulence had a high proport ion of PAS-resistant mutants , the presence of these mutants wo~ld on.ly be detected in those tests in which the inoculum was larger than usual, so that many o f i h e strains would appear to be similar in their sensitivity to the strains of high virulence.

If the two groups of strains were the descendants of organisms that had evolved in different regions or under other different circumstances (the larger group might, for instance, be strains that have been endemic in India over a long period and the smaller group be derived' f rom recently introduced strains of Western origin), the occurence of low virulence, high peroxide susceptibility and resistance to thiacetazone and PAS in the larger group might be the result of a series of causally unrelated evolutionary steps, rather than the product of some common mechanism which found its expression in each of these characters. In this connection, it is of interest that strain'; f rom Chinese patients in Hong Kong resemble those from Indian patients in their pattern of sensitivity to thiacetazone (Mitchison and Lloyd, 1964), yet they are similar to British strains in their virulence in the guinea-pig (Dickinson and others, 1963). These findings indicate that thiacetazone resistance and low guinea-pig virulence are not always associated together.

We are grateful to the nursing and medical staff at the Tuberculosis Chemotherapy Centre who arranged for the collection of sputum specimens from the Madras patients. We are also indebted to a number of physicians at British Chest Clinics who are mentioned individually by Mitchison and others (1961) for providing sputum specimens from their patients. Dr. D. W. Henderson kindly provided the facilities for animal experiements at Porton and Mr. J. Randles ably assisted in these experiments.

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Bull. WId. Hlth. Org., 2.5, 313. DICKINSON, J. M., EEl:FORD, M. J.. LLOYD, J., and M1TCHISON. D. A. (1963). Tubercle (Lond.), 44, 446. EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCIL THI~,CETAZONE/DIPHENYLTHIOUREA INVESTIGATION (1960).

Tubercle (Lond.), 41,399. EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCIL PRETREATMENT DRUG RESISTANCE REPORT (1963). Tubercle

( Lond.), 44, 393. FRtMODT-MOLLER, J. (.1957). Technical Report of the Scientific Advisory Board, p. 153. Indian Council of Medical

Re~.earch, New Dell~i. FRIMODT-MOLLER, J., MA1 HEW, K. T., & BARTON, R. M. (1956). In Proceedings ofthe Thirteenth Tuberculosis Workers'

Conference, p. 157. Tuberculosis Association of India, New Delhi. JENSEN, K. A. (1955). Bull. int. Un. Tuberc., 25, 89. MITCHiSON, D. A., BHATIA, A. L., RADHAKRISHNA, S., SELKON, J. B., SUBBAIAH, T. V., & WALLACE, J. G. (1961).

Bull. Wld. Hltlt. Org., 25, 285. MITCHISON, D. A.. & LLOYD, J. (1964). Tubercle (Lond.), 45, 360. MIrCHiSON, D. A., SELKON, J. B., & LL6YD, J. (1963). 3. Path. Bact., 86, 377. MlrCHISON, D. A., WALLACE, J. G., BHATIA, A. L., SELKON, J. B., SUBBAIAH, T. V., & LANCASTER, M. C. (1960).

Tubercle (Lond.). 41, 1. NAIR, C. N., MACKAY-SCOLLAY, E. M., RAMACHANDRAN, K., SELKON, J. B., TmPATHY, S. P., MITCHISON, D. A., &

DICKINSON, J. M. (1964). Tubercle (Lond.), 45, 345. SELKON, J. B., SUBBAIAH, T. V., BHATIA, A. L., RADHAKRISHNA, S.. & MITCmSON, D. A. (1960). Bull. Wid. Hith. Org.,

23, 599. SUBBAIAH, T. V., MITCHISON, D. A., & SELKON, J. B. (1960). Tubercle (Lond.), 41,323. THOMAS, K. L., JOSEPH, S., SUBBAIAH, T. V., 8,. SELKON, J. B. (1961). Bull. WId. Hlth. Org., 25, 747. TUBERCULOSIS CHEMOTHERAPY CENTRE, MADRAS (1960). Bull. Wld. Hlth. Org., 23, 535.