906

Methods and Devices

A RAPID TEST FOR BETA-LACTAMASEPRODUCTION BY HÆMOPHILUS

INFLUENZÆMARY P. E. SLACK D. B. WHELDON

D. C. TURK

Department of Bacteriology and Regional Public HealthLaboratory, Radcliffe Infirmary, Oxford OX2 6HE

Haemophilus influenzae type b is a common cause ofmeningitis and other acute life-threatening infections ofchildren which must be treated without delay. In the1950s chloramphenicol was the treatment of choice.Since the introduction of ampicillin in 1961 many clini-cians, especially in the U.S.A., have preferred to use thisdrug alone, because of fears of chloramphenicol toxicity.In 1974 ampicillin-resistant strains of H. influenzce typeb were reported’ and are now widely distributed. Thereis clearly a need for a test which rapidly establishes thetrue ampicillin sensitivity of strains of H. influenzae typeb isolated from clinical material. Ampicillin resistance inH. influenzx is almost always due to the production ofp-lactamase, intrinsic resistance being uncommon.3 3

Several methods for the detection of p-lactamase havebeen described.4-1O We describe a new rapid acidimetrictest for the detection of p-lactamase production by H. in-fluenzae, which uses strips of filter-paper impregnatedwith penicillin and a pH indicator. These strips may bestored for several months, ready for use when required.The test can be performed as soon as there is visiblegrowth on the primary culture plates. The principle ofthe method is that any p-lactamase present will hydro-lyse benzylpenicillin to form penicilloic acid, with a fallin pH which is demonstrated by a change in the colourof the indicator.

Preparation of Test Strips600 mg of unbuffered benzylpenicillin is dissolved in 4 ml

of 0-1% aqueous solution of bromocresol purple. 0.05 ml 0 1mol/l sodium hydroxide is added. Whatman’s no. 1 filter-

papers (9 cm diameter) are immersed in the solution and thenimmediately dried in a vacuum desiccator with active desic-cant. After drying, the paper is cut into strips approximately0.6 cmxl-2 cm. The strips are stored at 4°C with silica geland will retain their activity for several months.

Test

One drop of distilled water is placed on a clean microscopeslide and covered with a test-paper. The paper should be moistbut not oversaturated. A bacteriological loop is used to sweepacross the surface of a young culture of the isolate and is thenstreaked across one end of the strip. In older cultures, touchingone colony is enough, but because sometimes only some of thecolonies in a strain produce &bgr;-Iactamasell it is better to touchseveral colonies. If p-lactamase is produced the colour of thestreaked portion of the strip changes from purple to yellow in5-10 minutes. Non-&bgr;-lactamase-producing strains change thecolour of the strip, if at all, to a deeper purple. Known positiveand negative controls are applied to each strip. The test is easilyread. When dry the colours on test-papers remain for 24 hours.Results

44 strains of H. influenzae, all recently isolated frompatients, were tested for &bgr;-Iactamase production by thismethod and also by two methods in current use in our

laboratory-the chromogenic cephalosporin substratemethod’O and a modification of the Kjellander and Myr-bäch plate method.8 17 of the 44 strains were includedbecause they had given positive results on earlier routinetests with one of these methods. All 17 gave positiveresults with all three methods, whereas the remaining 27strains gave negative results by all three methods.Minimum inhibitory concentrations (M.l.c.) of ampi-

cillin (with carefully standardised inocula and multi-point inoculation on chocolate-agar) were also estimatedfor 33 of the 44 strains. 21 strains gave MJ.c.s lessthan 0.6 mg/l, and all these had given negative resultsin all three tests for p-lactamase production. Of theremaining 13, all of which gave an nt.i.c. of 2.5 mg/tormore, 12 had given positive results in all three &bgr;-lacta-mase tests. The thirteenth (with an M.l.c. of 20 mgll)had given negative results in all three tests. This strainwas therefore clearly an intrinsically resistant strain.

Thus there was total agreement between our methodand the others in the recognition of ampicillin resistancedue to p-lactamase production.Discussion

The test described here is simple and rapid, and relia-bly identifies j3-lactamase-producing strains of H. infl-uenzce. It may be performed as soon as there is anyvisible growth on the culture plates (5-6 hours), andrequires only a small number of organisms. This featurecould be of value in cases of partially treated meningitis,where there is often only a very scanty growth of thecausative organism. The test gives the clinician a morereliable indication of the response he can expect to ampi-cillin in infections caused by H. influenzae type b thanthat provided by antibiotic disc sensitivity testing. It isalso more rapid. The method may be used for routinesurveillance for &bgr;-Iactamase-producing isolates of H.

influence. Conventional antibiotic disc sensitivity test-ing of H. influenzce with ampicillin and chloramphenicolwill ensure the detection of the rare ampicillin-resistant,non-p-lactamase producing strains and also those resis-tant to chloramphenicol.Requests for reprints should be addressed to M.P.E.S.

REFERENCES

1. Gunn, B. A., Woodall, J. B., Jones, J. F., Thornsberry, C. Lancet, 1974, ii,845.

2. Tomeh, M. O., Starr, S. E., McGowan, J. E., Terry, P. M., Nahmias, A. J.,J. Am. med. Ass. 1974, 229, 295.

3. Thornsberry, C., Baker, C. N., Kirven, L. A., Swenson, J. M. Antimicrob.Agents Chemother. 1976, 9, 70.

4. Catlin, B. W. ibid. 1975, 7, 265.5. Jorgensen, J. H., Lee, J. C., Alexander, G. A. ibid. 1977, 11, 1087.6. Rosen, I. G., Jacobson, J., Rudderman, R. Appl. Microbiol. 1972, 23, 649.7. Escamilla, J. Antimicrob. Agents Chemother. 1976, 9, 196.8. Kjellander, J., Myrbäch, K. E. Acta path. microbiol. scand. 1964, 61, 4949. McGhie, D., Clarke, P. D., Johnson, T., Hutchison, J. G. P. J. clin. Path.

1977, 30, 585.10. O’Callaghan, C. H., Morris, A., Kirby, S., Shingler, A. H. Antimicrob.

Agents Chemother. 1972, 1, 283.11. Gray, B. M., Hubbell, C. A., Dillon, H. C. ibid. 1977, 11, 1021.

ADDENDUM

Two penicillin-resistant strains of Neisseria

gonorrhaeae, kindly supplied by Dr A. Percival, were alsotested for p-lactamase production using the method de-scribed above. Both strains gave well-defined and posi-tive results within 5 minutes. This suggests that themethod may also be used for the routine screening forpenicillinase production by gonococci isolated fromclinical material.