The susceptibility to hydrogen peroxide of Indian and British isoniazid-sensitive and isoniazid-resistant tubercle bacilli

7"ubrrclr, Land.. (1960), 4 I, 323

ORIG

I NAL

ARTICLES ii

i

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The Susceptibility to Hydrogen Peroxide of Indian and British Isoniazid-Sensitive and lsoniazid-Resistant Tubercle Bacilli By 'I'. V. S U B B A I A H , D. A. M I ' I ' C H I S O N ) and J. B. S L I , K ( ) N from Tiu' 7"uberculosis Chemotherapy Ce ,tre , 3ladras "

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Cohn and others (1954), Biinicke (195'I-) and Knox, M e a d o w and Worssam (1956) have sJiown that isoniazid-resistant, catahtse-negative strains of" tubercle i)aciili, whether obtained l)y in vitro selection or from treated patients, were inhibited by hydrogen l)eroxide at lower concentrations th;tl'l their isoniazid-sensitive, catalasepositive l)arent strMns. These authors also demonstrated that Ityth'ogen pcmxMe was more rapidly bactericidal to the catalase-negative thatt to tire catalase-positive strains. Kreis and l_.e ]oulNmx (1957a) dcseriimd a quantitative bactericidal test, in which a large inoculum (0.8 mg./ml.) of a dispersed culture was exposed to (I,03 pt:r ccitt w/v hydrogerl peroxide for 3 hrs. at 37~C., and a viabh, count ofthe surviving imctcrial population was then made. Strains composed of catalase-negative organisms were completely killed, whereas there was little fall in tilt" counts of'those with high catalase activity. Such a test shottld provide an estimate of the pro'portion ofcatalase-positivtorganisms in ;t strain composed o f a mixture of catalase-positivc arm c;ttalase-negative bacilli, l lowever, the ituthors showed that a small inoculum of catalase-l)ositive organisms was killed IW the prolonged contact with the peroxide employed in the test: ti,e reason for the survival of the bacterial populations in the large inocula of thcse organisms norrnally used was that they were capal)le of destroying muci~ c)t"tht" peroxidc itself during the pe,iod of exposure. Thus tire method could not i)c. rt:lied upo, r to detect the presence ofsmall numlmrs ofeatalase-positive Imcilll in a prt:domin;tntly catalase-negative strain, nor was it ce,'tain t() what extent any of tht." estimated proportions of thesc pol)ulations would correspond accurately with the true proport ions. No experiments were described in which the size of the inoculum, the concentration of peroxide or. the exposure period were varh.'d to obtain optimal conditions. T h e present work describes an attempt to) modify, tile method of Kreis and l.e .Joul)ioux (1957a) so that it wottld accurately estimate the relative prolmrtions o1" catalase-positivc and catalase-negative organisms ira strains containing mixtures of the two types. A bactericidal test was chosen in preference to a bacteriostatic test, since it is ditficult to obtain qtmntitative measurement witll the latter technique. In perfi)rming a bactericidal test residual peroxide must be inactivated or removed by dilution so that it does riot inhibit tim growtll of surviving organisms. Knox, M e a d o w and Worssam (1956) removed peroxide by centrifiJgation and washing, but this method was considered impracticable if this test were to be used on a large 1Now at the Medical Research tT)mncil's Unit fi)r Research on l ) r . g Sensitivity in TuberculcM~, l)ostgraduate Medical Schex)l of l.ondon. tUnder the joint auspices of the Indian Council of,Medical Research, the M:tdra~ State (]overnment, the World Health Organisation and the IIritish Medical Research Council.

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scale, and likely to produce inaccurate counts on the surviving organisms. In the present work the method of removal of peroxide ',','as studied as ,,.,,ell as the determinatit,n of th,: optimal per,)xide concentratitm and period of exposure which would kill all catalase-negativ,: ,~rganistns, but would leave catalase-positive organisms unallbcted. In addition, the method of Kreis & l_.e.Joubioux (1957a) was modified l~v reducing the inoculum of organisms exposed to peroxide so that catalase-positive I,~willi would not be able to dt'stroy peroxide during the test itself. The standardised I,actcricldai test was then employed in comparing the susceptibility to peroxide of isoniazid-sensitive strains fiom British and Indian patients, and in investigating the relationship between the peroxide susceptibility and the catalase activity of their iscmiazid-rt:sistant mutant strains. Materials and Methods

Strains qf tubercle bacilli Strain 1-137Rv had been maintained at the Postgraduate Medical School of l.ondcm on l_.6wenstein lensen medium fi)r over five years. This strain had a degree of catalasc activity normally encountered among other isoniazid-sensitive strains. Strain B, resistant to 50 /.tg/ml. i.,.'oniazid and catalase-negative, ,,,,'as obtained fi'om strain l i37Rv by in vitro selection in medium containing isoniazid (Barnett, Bushl U and M itchison, 1953). It had been maintained on isoniazid-fi'ee I_,6wenstein-.Jensen medium for the past five years. Strain BI v/as obtained by passaging strain B in 71110 mediuln containing 50 Fg/ml. isoniazid at the time of the experiments to be described. A t,)t;tl of" 17 isoniazid-sensitive strains was obtained fi'om 9 Indian and 8 British patients who had t~ot received anti-tuberctdosis chemotherapy, and heavy suspensions ~f tllest" strains were inoculated on to slopes o1"l..6wenstein.-.Jensen medium containing 0.2 and 5 Fg/ml. of isoniazid to obtain resistant, mutant colonies. The colonies,,','ere stlbctlltured and their catalase activities measured. The most catalase-positive of the lnutant clones fi'otn 0.2 /.tg/ml. isoniazid and the most catalase-negative of the mutant clones from 5 p.g/ml, isoniazid were selected for fi~rther study.

Media 1.6wenstein-lcnsen medium without potato starch (Jensen and Kiaer, 1959) was used for the maintenance of all cultures and isoniazid sensitivity tests (Medical Rest'arch Council, 1953: Tuberctdosis Chemotherapy Centre, 1959). 7I-I10 Tweenaibtlmin medium, as described by Cohn, M iddlebrook and Russell (1959), but with the omission of glycerol, was used for sul)cultiwltioll of strains in liquid medium. 71-110 oh:it acid-albtlmin medium (double strength), a modification of 71"I10 medium (Cohn, Micldlelwook & Rttsscll, 1959) was used for viable counts. The composition was: KH2PO.t, 2.0 g; Na:~1-11'O.vl2 1-1o.0, 12.6 g; 1-Na glutamate, 1.0 g; (N1-t.1)~ SO4, 1.0 g; Na citrate, 0.8 g; ferrous ammoniutn citrate (green), 160 mg; MgSO4. 7H.,0, 100 tng; CaCl,.,. 21-1,.,0, 1.0 rag; ZnSO a. 71-I20, 20 rag; CuSO 4. 5H.,O, 2.0 rag; pyridoxine HCI, 2.0 rag; biotin, 1.0 rag; glycerol, 4.0 ml; distilled water to I litre. The medium was autoclaved at 15 lb./sq, in. for 20 rain. and to it was added, with aseptic precautions, (a) 200 ml. of a solution containing 5 per certt bovine albumin (Fraction V, Armour l.,td.), 2% glucose and 0.06~,, oleic acid, which had been steriliscd by Seitz liltration and (b) 0.6 ml. ofcatalase 1 rng./ml., sterilised by filtration through a sintercd glass fiher (U.I;'. grade, Pyrex brand).

Reagents (I) lO'drotlen peroxide: 30~,,, w]v. 'Analar' (British Drug House). Solutions were standardised by titration with permanganate. ('2) Catalase: Crude beef liver extract (Nutritional Biochemical Inc., Ohio, U.S.A.).

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M/15 phosphate bu.[fer: pH 7.0, prepared according to S~,r,-nsens standard tal)les, and autoclaved at 15 lbs. tbr 20 rain.

Viable C:ount T h e silica gel method of Selkon and Mitchison (1957) was t,mphLved for x'ial)ic counts, raodified I)y the use of double strengtl: 71-I10 medium.

Catalase estimations Estimates of qualitative catalase activity ('i'ubercuh)sis C,hemotllerapy Centre, 1959) and semi-quantitative catalase activi.~y (Kreis, Le .](mlfioux and Pariente, 1956; Kreis & l..e .]ottbioux, 1957b) were carried out on all the strains.

7-he standard bactericidal test

T h e strain to be examin.:ct was grown for 8 ditys ill 7 H I 0 "l'ween-albumin liquid medium and 0.2 ml. volumes were added to I oz. screw capped I)ottlcs cont:tining (a) 3.8 ml. of 0.02 w/v hydrogen peroxide in pH7 phosph;lte bullk-r and (b) 3.1:1 nil. of buff'or alone. The bottles were inctilmted tbr 90 rain. at 37~ and viable counts were then set up fi'om each bottle, 0.2 ml. \'ohlmes of the l'eactiot~ SUsl)ensi()ns , and ()f 1:40 and 1:1600 dilutions being added to the viable count bottles, wlfich containecl 3.1 ml. doublc strength 7I-I10 mcctium. After addition of 3.3 ml. silica sol, the l)otlh's were incubated at 37~ for 4 weeks befi:we the COUlltS were read.

Experiments Delineating the Standard Bactericidal Test (1)

Termhmtion of the activity ( f t~drogen peroxide b), the addition qf catalaw

Preliminary experiments were carried out with the aim of developing a very simple method in which the organismscould be exposed to hydrogen peroxide in viablecount bottles. Strains H 3 7 R v or B were addecl to vi;tl)le-count bottles containing I i . i ii ' "/FIB37RV/Butfer ~ / Controls 8 __ I./BI -o /

H37 Rv Exposed to Peroxide

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Exposure period (rnin.) FiG. 1. - V i a b l e counts oll strain H 3 7 R v and its catala.se-negative variants, B and BI, during exp~rsurc to 0.02 per cent h y d r o g e n peroxide, 9 N o growth. 4,

326

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various concentrations of peroxide in 7 H I 0 double strength medium and, after incubation, catalase was added to destroy it. A concentration of 0,005 per cent H,00., had no bactericidal action on strain B, while, even ;tiler exposure for 80 rain., a sn~all proportion o1" this catalase-ncgative strain survived exposure to 0.0I per cent H,0,.. On the other hand strain H 3 7 R v fhiled to survive exposure to 0.0.5 per" cent H20 .. for five rain. With the intermediate concentration of 0.02 per cent l-l:~0~, strain B was completely killed, while viable counts on H 3 7 R v werc almost unafl'ected. "rile termination of the activity of the peroxide by catalase was, however, unreliable, since addition of`catalase 30 rain. before the inoculum of strain B to medium containing ;is low a conccntratiorl ;is 0.02 per cent H.,02 did not always allow growth of the organisms.

(2)

7~mtination of the activity of hydrogen peroxide to, dilution

In the remaining experiments the test strains were incubated with 0.02 per cent H._.0~ ira pH7 buflbr solution its described for" the standar.d bactericidal test, and the activity of the peroxide' was terminated by dilution (1:15 or more) when 0.2 ml. of the suspension was added to the m e d i u m in the viable count bottles or to dilution tubes. T h e results of ir~cubating strains H37P, v, B and BI with peroxide for various periods ave shown in Fig. 1. There was no change in the viable counts on strain H 3 7 R v during 60 rain. exposure to peroxide, though a fall fi'orn 7.8 to 6.5 log 1() viable units/ nil. occurred between 60 and 180 rain. On the other hand no sur~;iving organisms of strain B1 were detected at or after" 60 min. exposure to peroxide. In an earlier experiment, performed ira a similar manner, strain B continued to yield a small ntlmber of colonies after exposure to 0.02 per cent peroxide for as long as 90 ;~nd 120 rain. Sub-culttwes fi'om these colonies showed definite and sometimes high catalase activity. Reverse mutation fi'om catalase-negativity to catalase-positivity may have occurred ira strain B during the 5 ),eat" period of maintenance on drug-fi'ee medium since its initial production fi'o:n strain H 3 7 R v . In view of this possibility, it was passaged through T w e e n - a l b u m i n liquid m e d i u m containing 50t.~g]ml. isoniazid and the resulting strain (B1) was employed ira further experiments. T h e slower killing ofstrain B than of strain BI, illustrated in Fig. 1, suggests that catalasepositive reverse-rnutants were probably eliminated during tile passage. (3)

Reconstruction experiments

Mixtures containing known proportions of isoniazid-sensitive, catalase-positive strains and their resistant, catalase-negative, variant strains were exposed to 0.02 per c'ent peroxide for" various lengths of time to see how accurately the bactericidal test would estilnate the proportion of" catalase-positive organisms in the mixture. T h e resttits of two experiments with strain H 3 7 R v and its catalase-negative variant Bl, and with an Indian isoniazid-sensitive strain 1522.1-S and its catalase-negative variant 15221-R are set out in Tables I and If. I n column (a) of these tables arc shown the control viable counts on the strains exposed only to buffer, and in columns (b), (c) and (d) are the counts found after exposure to peroxide. Ira both experiments little change had occurred in the counts on the parent eatalase-positive strains after" 90 rain. exposure to peroxide, whereas the counts on the catalase-negative variants had been reduced fi-om over 107 to less than 102 viable units]ml, in the same period. T h e n u m b e r of catalase-positive organisms to be expected in the mixtures, calculated fi'om the counts on the buffer controls on the assumption that all catalase-positive organisms would survive and all catalase negative organisms would be killed, are given in column (e). Comparison of the values in column ( e ) w i t h those ira columns (b), (c) and (d) shows that the expected counts agree well with those found after exposure of the mixtures to peroxide for 90 rain., though they are on average 36 per" cent lower. Exposure for 120 rain. yielded estimates that were even lower, presumably due to greater killing of the catalase-positive organisms by the peroxide.

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Composition of strait, teJted (volume per cent)

Found

Buffer H37Rv

only mill. (~i"

BI 0 I00 90 99 99 "9

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Periods of 45 rain. or 60 min. exposure yielded high estimates when t~e F,rolmrtion of catalase-positive organisms in the mixture was very haw (0.001 per cent), presumably due to survival of a small proportion of the catalasc-negative bacilli. T h e opti,nal period of exposure therefore appearect to be 90 rnin. and this was adopted fiw further work. In column (f) is set out the counts to be expected in the mixtures after exl)osurc to peroxide for 90 rain. calculated fi'om tlw count on the l)ure catalase-p,,sitivc strain after exposure to peroxide for the same time and on the assmnl.)tion that all catalase-negative organisms had been killed. T h e Counts in column (f) agree very closely with those found on the mixtures (Table I, column (d), and "l'ai.)lc II, column (c)). It is cleat" that, even when the mixtures contained as high a proportion as 10 per cent of catalase-positive organisms, the bactericidal activity of the l)croxidc ,,,,'as not diminished. Thus, the test can be relied upon to estimate accurately the proportion of catalase-positive organisms in a mixed l)opulation over a very wichrange of mixtures. It is also capable of detecting very small nurnbt:rs (! in l0 s) of catalase-positive organisms in a predominantly eatalase-mzgative culture. "l',xm.v. I I.

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~[IXTURES O F CATAI.ASE-|)osrrwv: AND CAIAI.AMJ.NEGATIVE INDIAN ~TRAINS

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Found

Composition of strain tested (volume per cent)

l-gposed to [ After exposbuffer i me to tl~O ~ on/)' [ f o r ~90 m.n. .

Period of exposure to lifO=

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129 63 0.10

7,88

7.51 7.80 7.47 7.7O

.Ill 37 5.1 66 65 32

0.25

46 2.1 1.3

Results ] S()NI/tZJ | ) - ~ E, NSIT! %'E ~~162 l NS

In "l'abh" 111 are set out tile results of tile standard, bactericidal, peroxide sensitivity test on II llritish and 7 Indian isoniazid-sensitive, catalase-positive strains. The percent;tge of the f,'ganisms in the strain that survived exposure to 0.02 per cent peroxide Ii,r 90 wi,:. is shown in the right hand column. A m o n g the British strains the average percentage was 67 (ra~ige of 32% to 126~,,), a value that agrees well with the iwrcenmges of survivors (64 %) in similar isoniazid-sensitive strains obtained ira the reconstruction experhnents. In contrast, 4 of the Indian strains appeared rather more suscel)tibh, to peroxidc. 'l'he average percentage of surviving organisms was 35, and the range t)t"(1.10 % to 129 % was wider. T h e difference in susceptibility between lhitish and Indian strains attains significance at the 5% level (l"isher-Behrens test on logarithmic ,l~,cragcs). ISON|AZH)-Rl~ts'rAwr S'rRAtNS 'l'l,' results c~f the standard hydrogen peroxide sensitivity test on 24 isoniazid-resistant strains, all ~fwhich were chines derived from single colonies, arc set out in Tal)le IV, iu which the strains are arranged in order of their eatalase activity. Briti.di S t r a i m

C~,nsidering first the results or, the 13 British strains, 6 of these (1952b to 1919b), witll semi-quantitative catalase activity of 0~,,, to 10% and no activity in the qualitative lest, all contained populations of bacilli apparently entirely susceptible to p~!ro.xide, the percentages of surviving organisms beir,g less than 0.001 ~ . The rema,n,ng 7 strains with relatively high degrees of catalase activity in the semiquantitative test (10~',, to 50'~',,) and in the qualitative test (1-plus or more) were all moderately resistant to peroxide; the percentages of surviving organisms ranged from 0.11 ~,, to 23 %. These values were lower than any of those of their parent isoniazidsensitive strains ('I'al)h" 11 I ) . T h e degree of resistance to isoniazid of the strains has been expressed in two ways. "l'he tirst of these was the minimal concentration of isoniazid inhibiting the growth c~f 2(1 or more colonies (Table IV, column (b)); the resuhs showed only a weak

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association w i t h the, c a l a l a s e a c t i v i t y or h y d r o g e n

p e r o x i d e s u s c e p t i l J i l i t y c)f" the strairi.

The secolld way (Table l\", coJtlnln (a)) was thc highest COilcentratioll oJ" isoniazid whicll allowed growth of at least innumerable cliscrete colonies (the control drug-free slopes yielded conflueilt growth). Such resiilts giw: a better measure of tile degree of resistance of the majority of the i)acterial populations iil the resistant clones and ;ire h.'ss afl'cctecl by the presence of rare organisms with ;i.n exceptionally high degree ol" resistance. As me;tstirccl in this way, all of the 7 lh'itish strains with inoderate resistance to hydrogen peroxide and relatively high catalase activity had low degrees of resista n t e v,,ith growth oil slopc.'s containing up to but not more than .5 izg.]ml, isoniazicl, whereas the remaining 6 strains with peroxide-susceptible poplihitions and low catahlse activity all grew oil 50 v.g./ml, isoniazid. hldian Strains

Considering the I1 Indian isoniazid-resistant strains, 3 had no delnonstrabh" catalase activity in both the serrii-quailtitative and tim qualitative tests and tlicir bacterial polmlatiolis appl'ared entirely suscel)tible to" peroxide. "l'lie other II strains had from 3().o/,, to 90 % activity in the semi-quantitative test and at least l-plus activity in the qualitatiw: test. Amorig these ~; strains, .5 had a peroxide susceptilli/ity coinpar;lille with the British isoniazid-resistant strairis wliich retained a relatively high c;ltahisc activity; the percentages of peroxide-resistant organisms ranged ti'orri 0.19 % 1o 22,Y,,. However, the remaining 3 strains (31641Ja, 3 l134{~,lJand .31370a) appeared considerably more susceptillle to peroxide; the percentages of peroxide-resistant ~lrgaiiislils were < 0.00005 9',,,, < 0.()f)5')',,, and 0.0111 ~ , respectively. 'l';lble 1\ 1 ;iiso shows all associ;~tion between the degree of isoniazid rcsistatice and the catahtse activity of" lildiarl strains, I>ut this is somewhat less marked titan ;inlollg the lh-itish strains. "l'he isoliiazid-sensitivity of strains 3164Via, 31(;41111 iiiid Dl137()a did not cliffvr from that of the remaining strains with siinilar degrces of catalase activity. The peroxide stisceptibilitics of pairs of the parent isoniazicl-sciisitive strains and their isoni;izid-resistant m u t a n t strains are cornpared in Table V lbr those isoniazkl'I'AIII.I" l l t . - 'rllp. [['VI)R()I';F.N I'ERr ,~[L~CIS.I"I'IIIII.I'I'Y Ill' IM)NIAZIII-~ENM'F1VE P A R E N T ~'I'RAINS AND "l'llla.llt ('.ATA I.hS V.-] ~O.SIl'l V IS.,

[ Sf).MIA Z I [)- l{lk%lS |'A N'rl .~'i'RA [N.S

,'~1 U"I'ANT

Peroxide reJiatmt organLrms (per cent) St ra in ,'lb.

0 r i.4,in

~f Jttain

Briti.sli

I [)52 I il29 I 959

,,

,, ,, ,,

I 907 i 1107

Indian .,

305 lO

3"t25.t 3980-! 'l(g)38

3'1024

31370

~emitive parent strain

Eoniazid.

I

rr~iManl mutant Jtrain

66 G5

tO

32

2.1 23 13

0.11

54 -18

63

:

46

,,

I "3

i : ;i

,, ,,

0"25

i

,.

;

h on in.: ill.

2" I

0"10

{).37 22 I "5

0.98 O' 19 0018

SI.IS(.:It, I ' T I i I l i , I T Y

'1"(} I I Y I J I " , ( I ( ; I t N

I'I';R~[IXil)I".

3."11

resista,~t strMns that still retained at least 10'~,,, catalast: activity in the setniquantitative test and l-plus activity in tim clualitative test, and xvller~..9 tests on Imth strains of the pair were availaiJle, it ,,viii be appreciat,:d that tht.-.se results are. selected from 'l'ablcs Ill and IV. l:cn- tire .5 pairs of British slr;tlns no associatic,n is apparent between the peroxide susceptibilities of the isoniazid-sensitive and resistant strains. A m o n g the t3 pairs of Indian strains there is a suggeslicm that ;i low perct:ntage of peroxide-resistant organisms in the parent suairr may Im associated with it lmv percentage in the isoniazid-resistant m u t a n t strain. 'i'his associatior~ fails t~ atttain statistical significance (P---.0,1-0.05), ba, t it is noteworthy that the isoniazid-scnsitivtparent strain 31370 had the lowes't percentage of per,xide-res'istant cn'ganisl,rs a m o n g ;ill of the isoniazid-sensitive strains, and its isoniazid-rc~istant mutant strain also had a pt:rcentage of peroxide resistant orgarfisms lmvcr than was fimnd in any of the British strains or of the other Indian strains.

Discussion T h e hydrogen peroxide.bactericidal test described above has overcome tim nlail~ defects of the lnetluM of Kreis and 1.e .Joubioux (1957a), namely the use of a very large inoculum in which catalase-positive organisms were. resprmsil)h" lb," destructi,m of the peroxide dr,ring tim test exposure, and the corrsequent use of a l)rolotlge:t exposure period. Ira tire present test both tire size of tire irmculum and the exposure period have been reduced, so that it has been possible to trstimatt: accurately the proportion of isoniazid-sensitive, catalase-positive bacilli in a mixture of c;ttala.'.'t'positive and catalase-negative organisms. T h e a c c u r a c y w a s lnaintaint:d over a wide range of proportions of tim two types r)t"c)rgani.,'ms and tim test was capabh: of detecting catalase-positive bacilli, even v,,hen these were present as only 1 ira 10:' (,f the bacterial l)opulation. "l'he dilution of tire hydrog(:n perr)xide ira the vidbh" cr)unt procedure appeared to l)e sufficient to allmv sul)sequent growth of any organisms wllic}, hacl survived the period of exposurt: in tim test. T h e counts in the dit'li:rent dilutions from ;t single test showed good proportionality. On the other hand, preliminary attempts to termin;tte the activity of peroxide lay addition of catalase ',,.'ere uttsuccessful, when the exposure to peroxide and the viable c o u n t were carried o u t in tim same Imtth;. Sui)sequent inhibition of growtll may have hcen duc to the high concentration of nascent oxygen ira tire culture medium or to the inactivati~m of tim catalase l~y the peroxMe before it had reduced the peroxide concentration to a sulticiently low vahte to allow growth. British and Indian strains showed difli:rences in their susceptibility to hydr~,gen peroxide. A m o n g tire isoniazid-sensitive strains, all of which were inhibited by 0.2 ~tg/ml. isoniazid, the 8 British strains had a fiiirly uniform high degree of resistance to 0.02 per cent peroxide, whereas 4 of tim 7 Indian strains were more susceptible. No difference was apparent in tim catalase activity of the strains from the two countries. A similar difference v,'as also apparent a m o n g those isoniazidresistant strains which retained catalase activity. T h e 7 British strains contained at least 0.11 pet- cent peroxide-resistant organisms, whereas 3 of the 8 Indian strains contained 0.018 per cent or less of these organisms. "l'he Indian strains had a degree ofcatalase ;activity at least as high as that of the Fh'itish strains. T h e r e was a suggestion that isoniazid-sensitive Indian strains with increased susceptibility to peroxm~ yielded catalase-positive, isoniazid-resistant, m u t a n t strains also with increased susceptibility. A peroxide susceptibility lower than would be expected from the catalase activity in a proportion of Indian strains can b,: explained in various ways. T h e permeability of the cell wall of these organisms to h~'drogen peroxide might be lower than in the remaining Indian strains and ira those ti'om Britain. Winder (19~')) "

t~qj

332

"runv.RCLE

has shown that the cells of BCG are less permeable to hydrogen peroxide than those of M),cobacterium smegmatis. Alternatively, the Indian strains might differ in their proportions of intracellular and extracellular catalase. Finally there could be differences in tile biological systems affected by hydrogen peroxide within the cells. The relationship between peroxide susceptibility and animal virulence of Indian strains is being investigated. This is of particular interest, since Mitchison and others (1960) have shown that isoniazid-sensitive Indian strains of tubercle bacilli, of similar origin to those reported on here, had a wide range of virulence in the guineapig and were fi'equently less virulent than British strains. The composition of the surfhce of the bacterial cell is likely to play a major role in determining virulence (Bloch, 1950; Middlebrook, Coleman and Schaefer, 1959), and may also control the permeability of the cell wall to peroxide or to bacterial catalase. Furthermore, Coleman and Middlebrook (1956) have postulated that the diminished virulence ofisoniazid-resistant tubercle bacilli may be directly due to their increased suscep.tibility to low concentrations of hydrogen peroxide produced from glutathione m host macrophages. Such a mechanism could also explain the attenuation of a proportion of isoniazid-sensitive Indian strains. The occurence in a catalase-negative strain of reverse-mutation in vitro to catalase positivity appears to have been demonstrated during the investigations carried out on strain B. This strain was selected from strain HS7Rv in 195:3, when it was passaged 5 times through liquid medium containing 50 v.g/ml, isoniazi A It must then have been composed entirely of organisms resistant to 50 vtg/ml, isoniazid and catalasenegative. During the next 5 )'ears it was maintained by serial subcultivation on drugfree LOwenstein-Jensen medium. In the experiments reported here, colonies with full catalase activity were obtained from strain B after exposure to peroxide for 60 or more minutes. It seems reasonable to suggest that these colonies arose from catalase-positive organisms which had emerged by reverse-mutation during subcultivation of strain B on drug-free medium. Such reverse-mutants cannot have recovered tile full biological potential of the parent HSTRv organisms, since the)" were present as only a very small proportion of a population that was still predominantly catalase-negative. Evidence for reverse-mutation in stabilized isoniazid-resistant, catalase-negative strains of tubercle bacilli in vitro has been presented by Middlebrook (1958) and in monkeys by Schimdt and others (1958).

Summary A standard, bactericidal, hydrogen peroxide sensitivity test was developed, in which a viable count was done on strains that had been exposed to 0.02 per cent hydrogen peroxide for 90 rain. at 37~ This test h a s been found to estimate accurately the proportion of catalase-positive, ison]azid-sensitive organisms in a mixture with catalase-negative, isoniazid-resistant organisms. Among strains from British patients, 8 with full sensitivity to isoniazid were uniformily resistant to peroxide and 7, which were isoniazid-resistant but still retained some catalase activity, were more susceptible to peroxide, but contained at least 0.11% peroxide-resistant organisms. In contrast, among strains from Indian patients, 4 of the 7 isoniazid-sensitive, catalase-positive strains were more susceptible to peroxide than any of the British strains and 3 of the 8 isoniazid-resistant, catalasepositive strains contained 0 . 0 1 8 ~ or less peroxide-resistant organisms. All of tile 9 catalase-negative, isoniazid-resistant strains from both British and Indian patients were completely susceptible to peroxide. An association was found between low catalase activity, high susceptibility to peroxide and a high degree of resistance to isoniazid. This association was more clear cut among British than among Indian strains.

SUSCI~.I, T I I t I I . I T Y TO I{~'I)ROGEN I'I-'.ROXIDE

333

[~cf2re~ces Barnett, M., Bushby, S. R. M., and Mitchison, D. A. (19331 Brit..J. exp. Path., 34,568. Bloch. H. (1950) J. exp. Med., 91, 197. B6nicke, R. (19.54.) Naturwissertschaften, 41, "377. Cohn, M. L., Kovitz, C., Oda, U., and Middlebrook. G. (1934) ..liner. Rev. Tuberc., 70, 6 t l . Cohn, M. L., Middlebrook, G., and Ru.s.sell, W. F. (1957).7. olin. Im~sl., 38, 13-t9. Coleman, C. M., and Middlebrook, G. (19.56) ,.liner. Rev. Tuberc. 74, 42. .]'ensen, K. A., and Kiaer, I. (1959) Bull int. Un. Tuberc.. 29, 82. Knox, R., Meadow, P. M., and Worssam, A. R. H. (19.56) ,.liner. Rev. Tuberc.. 73, 726. Kreis, B., and Le Joubioux, E. (1957a) ..Inn. lnat. Pasteur, 92, 123. Kreis, B., anct l.ejoubioux, 1'~.(1957b) Rev.franf. el. din biol., 2, 4-t7. Kreis, 13.. Lejoubioux, E., and Pariente, D. (19.56) ,.Inn. Inst. Pasteur, 91,932. Medical Rest-arch Council (1953) Lancet, ii, 213. Middlebrook, G. (1958) Transactions of the 17th conference on the chemotherapy of tulx-rculosis, p. 261. Veterans Administration, Washington. Middicbrook, G., Coleman, C. ,M., and Schaefer, W. 13. (19.59) Proc. nal..-lead. Sci., limb., 45, 1801. Mitchison, I). A., Wallace, J. G., Bhatia, A. L., Selkon. J. 13.. Subbaiah, T. V., and l.ancaster, .M.C. (1960) Tubercle, Lond., 41, !. Schmidt, L. H., Grover, A. A., Hoffman, R., Rehm,J., and Sullivan, R. (1958)"l'ransacticms ofthe 17tli conference on the chemotherapy of tuberculosis, p. 269. Veterans Administration, Washingto,~. Selkon, J. 13., and Mitchison, D. t.k. (19.57) .J..~en. Microbiol. 16, 229. Tuberculosis Chemotherapy Centre (1959) Bull. lVorM Hlth. Or~.. 21, 51. Winder, F. {1960) Amer. Rev. Tuberc., 81,68.