931

In human beings fucidin is well absorbed after oraladministration. The drug tends to accumulate in theblood after repeated doses. It is excreted in the faeces butnot in the urine. The drug may cause slight gastro-intestinal upset. Systemic side-effects have not beenobserved.

REFERENCES

Allinger, N. L., Coke, J. L. (1961) J. org. Chem. 26, 4522.Baird, B. M., Halsall, T. G., Jones, E. R. H., Lowe, G. (1961) Proc. Chem.

Soc. 257.Godtfredsen, W. O., Jahnsen, S., Lorck, H., Roholt, K., Tybring, L.

(1962) Nature, Lond. 193, 987.

ANTIBACTERIAL ACTIVITY IN VITROOF FUCIDIN

MARY BARBERM.D. Lond.

PAMELA M. WATERWORTH *F.I.M.L.T.

From the Department of Bacteriology,Postgraduate Medical School, London, W.12

"Working with a full-time grant from the Medical Research Council.

Bacteriostatic ActivityTHE bacteriostatic activity of ’ Fucidin ’ for a variety of

gram-positive and gram-negative cocci was tested by serialdilution in solid medium. The results obtained with asmall inoculum (1 mm. loopful of a 1-in-500 dilution ofan overnight broth culture) are shown in the accompanyingtable.

Effect of serum.-In the presence of 10% serum theactivity was reduced approximately 16-fold, and with 50%the reduction was 64-fold.

Effect of pH.-In tests with 4 strains of Staphylococcusaureus it was shown that the activity was considerablyreduced in alkaline medium. Thus the minimum in-

hibitory concentration for all strains was: 0-03 at pH 6-0,0-06 at pH 7-0, and 0-25 at pH 8-0. The satellitismreferred to by Hilson (1962) is probably due to the factthat most bacteria tend to render nutrient agar alkaline,since similar rings of growth occur round agar cupscontaining alkali, made in nutrient agar plates containingbacteriostatic concentrations of fucidin.

Bactericidal Action

The bactericidal action of fucidin was tested againstStaph. aureus. Early experiments with the ’ Cellophane ’transfer method (Chabbert 1953) indicated high bacteri-cidal activity, but these results were subsequently shownto be due to a very high degree of carry-over of fucidin inthe cellophane. Subsequent tests were, therefore, carriedout by making surface viable counts.In tests with a small inoculum, giving approximately

5000 staphylococci per ml., a concentration of 0-5 g. perml. fucidin killed 90% of the cells in 15 hours and

practically sterilised the culture in 24 hours. With a

larger inoculum to give 3 million cells per ml. a concentra-tion of 0-5 Vg. per ml. also killed 90% in 15 hours, but thefew surviving cells subsequently multiplied, so that by30 hours visible growth had occurred. These organismswere shown to have an increased resistance to fucidin.The killing effect was similar with higher concentrations(5 and 25 pLg. per ml.) of fucidin, although with suchconcentrations growth did not occur later.

Development of ResistanceAs indicated above, when a large inoculum of a strain of

Staph. aureus is added to a concentration of fucidin whichis completely bactericidal for a small inoculum a few cells

BACTERIOSTATIC ACTIVITY OF FUCIDIN

* 39 strains penicillinase-producing.t Penicillin-resistant.

survive and later multiply. The minimum inhibitoryconcentration of such cultures has been shown to be 64or more times greater than that of typical cultures. Thusany large inoculum of staphylococci appears to contain afew mutant cells, which show a considerable degree ofresistance.When a strain of Staph. aureus is passaged in sub-

inhibitory concentrations of fucidin, grossly resistant cellsare less readily obtained. This is probably because themore sensitive cells not only greatly outnumber theresistant mutants, but also grow more rapidly.The addition of penicillin did not prevent the emergence

of resistant cells unless sufficient was added to cause

complete inhibition (see below).Synergy with Penicillin against Penicillinase-producing

Strains of Staph. aureusUnder certain conditions fucidin and penicillin can be

shown to act synergistically against penicillinase-producingstrains of Staph. aureus. The synergy is only seen with alarge inoculum and the extent varies greatly with differentstrains and appears to depend on the rate with which thestaphylococcus can destroy penicillin, so that relativelylittle synergy can be demonstrated with highly activepenicillinase-producers.

Thus, in a typical experiment with a weak penicillinase-producing strain, it was found that a large inoculum,which was only inhibited by 1-2 g. per ml. of fucidin byitself, was completely inhibited by the following combina-tions in g. per ml.:

On the other hand, with a rapid penicillinase-producingstrain 8 g. per ml. of penicillin had to be added to 0-25;jt.g. per ml. of fucidin to inhibit growth for 24 hours andeven in this mixture growth occurred at 48 hours.In further experiments it has been shown that the

inhibition of growth by the mixtures of fucidin and peni-cillin depends on delaying penicillin destruction. Thus forcomplete inhibition a mixture must contain enoughfucidin to inhibit multiplication of the test strain suffi-ciently to delay destruction of the penicillin in the mixturefor 2 to 4 hours. Such a mixture is bactericidal as well asbacteriostatic even for a very large inoculum, presumablybecause any fucidin-resistant mutants are killed by thepenicillin.

Comparison with Cephalosporin P 1A comparison of these results with those of Garrod and

Waterworth (1956) shows many similarities betweenfucidin and cephalosporin Pl. Both antibiotics have a

932

similar antibacterial spectrum, although fucidin is themore active; both show reduced activity in alkaline

medium, and with both antibiotics drug-resistant mutantsof Staph. aureus are frequent.Our investigation has shown that the combination of

penicillin and cephalosporin P is synergistic in a similarway to that of penicillin and fucidin; and there is cross-resistance between fucidin and cephalosporin Pl. Thusfucidin-resistant mutants isolated from 6 strains of

Staph. aureus by single step or repeated transfer in fucidinall showed a comparable increase in resistance to

cephalosporin Pl.Summary and Conclusions

Fucidin inhibits the growth of most strains of Staph.aureus, whether penicillin-resistant or not, in highdilutions, but most large inocula of staphylococci containresistant mutants.

A combination of penicillin and fucidin is synergisticfor many strains of penicillinase-producing Staph. aureusand the mixture is bactericidal even when tested with a

large inoculum.Attention is drawn to the similarity of fucidin and

cephalosporin Pi, but the antibacterial activity of fucidin isgreater.Our thanks are due to Leo Laboratories for a supply of fucidin and

to Prof. L. P. Garrod for a small sample of cephalosporin Pi, originallygiven to him by Dr. E. P. Abraham.

REFERENCES

Chabbert, Y.. (1953) Ann. Inst. Pasteur, 84, 345.Garrod, L. P., Waterworth, P. M. (1956) Brit. med. J. ii, 61.Hilson, G. R. F. (1962) Lancet, i, 932.

IN-VITRO STUDIES

OF A NEW ANTIBIOTIC (FUCIDIN)G. R. F. HILSON

M.D. Lond.

READER IN BACTERIOLOGY,ST. GEORGE’S HOSPITAL MEDICAL SCHOOL, LONDON, S.W.1

’ Fucidin ’ (ZN 6) is a steroid with antimicrobialactivity, and it has been available in the form of thesodium salt, a freely soluble white powder. The work

reported here was carried out with strains of Staphylococcuspyogenes and one strain of tubercle bacillus. Only thebroad outline of the techniques used and of the resultsobtained are described here; it is hoped to publish adetailed report elsewhere.

Stability of Fucidin in Bacteriological MediaA stock solution of fucidin (10 mg. per ml.) was prepared in

distilled water and sterilised by Seitz filtration. When storedat -20°C it did not apparently deteriorate over several months.In more dilute aqueous solutions, however, a slow loss of anti-bacterial activity was detectable. The stability of the drug innutrient agar (pH 7-4) and in Kirschner medium was investi-gated by inoculating and reinoculating these media daily withthe Oxford staphylococcus during incubation at 370C until theactivity of the drug had fallen below the inhibitory level, asindicated by the appearance of growth on the following day.By using different initial concentrations, and noting the numberof days before growth appeared on each concentration, anapproximate estimate of the half-life of the drug underdifferent conditions could be calculated.

In sterile nutrient agar at concentrations of 0-8 fLg. per ml. ormore, and in Kirschner medium at 0-1 fLg. per ml. or more, thedrug was quite stable over 4 weeks at 37°C. In sterile agar atlower concentrations it degraded slowly, with a half-life ofabout 8 days. However, in nutrient agar carrying surfacegrowths of staphylococci resistant to the concentrations tested

(0-1-3.0 g. per ml.), deterioration was much faster, with ahalf-life of about 1-5 days. The reason for this is discussedbelow.

Inhibitory ActivityStaphylococcus pyogenesThe minimum inhibitory concentration (M.I.C.) of

fucidin for different-sized inocula of 4 strains of

coagulase-positive staphylococci was estimated by carryingout surface viable counts (Miles and Misra 1938) fromovernight broth cultures on plates of nutrient agar

containing different concentrations (at twofold intervals),and on control drug-free medium, and comparing thenumbers of colonies and appearance of growth at dailyintervals for 10 days. The persistence of activity of thedrug in such plates was checked by daily reinoculationwith the Oxford staphylococcus, as described above.The nt.i.c. for inocula of fewer than 104 cocci, spread

over an area of about 2 sq. cm., was 0-025-0-05 g. per ml.,shown by very slow growth of individual colonies, thoughthese ultimately reached numbers equal to the correspond-ing control. On 0-1 g. per ml., no growth developedfrom these small inocula. For inocula of up to a fewmillion, each tenfold increase in inoculum requiredroughly a twofold increase in drug concentration to effectsuppression of growth at 2 days-i.e., there was a striking" inoculum effect ".On the areas receiving inocula of more than 104 cocci on

plates with more than 0-1 g. per ml. fucidin, discretecolonies of normal size could be seen at 24 hours, whichwere later shown to be resistant variants: the numericalincidence of these is reported below. Another phenomenonnoted at this stage was that in the neighbourhood of suchresistant colonies there was enhancement of growth ofcolonies partly suppressed by the drug-an appearancesuperficially similar to that usually known as " satel-litism ", and referred to below under that name.A survey of the fucidin-sensitivity of 240 strains of

staphylococci freshly isolated from patients (mainly frompus swabs, sputa, and urine) was made by streakinginocula of about 104 to 105 organisms on the surface ofnutrient agar plates containing fucidin 0’1 {ig. per ml.,with appropriate controls, and incubating at 37°C for2 days. All 240 strains were sensitive to this concentration,although 64 of them developed one or more apparentlyresistant colonies after 24 or 48 hours’ incubation.

Mycobacterium tuberculosisThe M.I.C. of fucidin for the H37Rv strain of human

tubercle bacillus was determined by titration in semi-solidKirschner medium (Knox 1955), using doubling dilutionsof the drug between 100 and 0-4 g. per ml. Two sizes ofinoculum were used, one consisting of 0-02 ml. of a 10-dayDubos culture added to each tube of medium (volume3-0 ml.), and the other was a similar inoculum from a1/100 dilution of the culture. By comparing the growth indrug-containing medium with that in controls at weeklyintervals up to 8 weeks, the M.l.c. was found to be 3-0 g.per ml., with a rather indeterminate end-point, due to thepresence of fairly large numbers of discrete colonies whichwere present where general growth was suppressed; thesewere fewer with increasing drug concentration, only twoor three occurring in the presence of 100 g. per ml. Itseems likely that these also were resistant variants, buttheir exact nature has not so far been investigated.

Bactericidal Effect on Staph. pyogenesThis was tested by using a modification of the Cello-

phane ’-disc transfer procedure of Chabbert (1953).