906
Methods and Devices A RAPID TEST FOR BETA-LACTAMASE PRODUCTION BY HÆMOPHILUS
INFLUENZÆ D. B. WHELDON MARY P. E. SLACK D. C. TURK
Department of Bacteriology and Regional Public Health Laboratory, Radcliffe Infirmary, Oxford OX2 6HE type b is a common cause of and other acute meningitis life-threatening infections of children which must be treated without delay. In the 1950s chloramphenicol was the treatment of choice. Since the introduction of ampicillin in 1961 many clinicians, especially in the U.S.A., have preferred to use this drug alone, because of fears of chloramphenicol toxicity. In 1974 ampicillin-resistant strains of H. influenzce type b were reported’and are now widely distributed. There is clearly a need for a test which rapidly establishes the true ampicillin sensitivity of strains of H. influenzae type b isolated from clinical material. Ampicillin resistance in H. influenzx is almost always due to the production of p-lactamase, intrinsic resistance being uncommon.33 Several methods for the detection of p-lactamase have been described.4-1O We describe a new rapid acidimetric test for the detection of p-lactamase production by H. influenzae, which uses strips of filter-paper impregnated with penicillin and a pH indicator. These strips may be stored for several months, ready for use when required. The test can be performed as soon as there is visible growth on the primary culture plates. The principle of the method is that any p-lactamase present will hydrolyse benzylpenicillin to form penicilloic acid, with a fall in pH which is demonstrated by a change in the colour of the indicator.
Haemophilus influenzae
Preparation of Test Strips 600 mg of unbuffered benzylpenicillin is dissolved in 4 ml of 0-1% aqueous solution of bromocresol purple. 0.05 ml 0 1 mol/l sodium hydroxide is added. Whatman’s no. 1 filterpapers (9 cm diameter) are immersed in the solution and then immediately dried in a vacuum desiccator with active desiccant. After drying, the paper is cut into strips approximately 0.6cmxl-2cm. The strips are stored at 4°C with silica gel and will retain their activity for several months.
Test One drop of distilled water is placed on a clean microscope slide and covered with a test-paper. The paper should be moist but not oversaturated. A bacteriological loop is used to sweep across the surface of a young culture of the isolate and is then streaked across one end of the strip. In older cultures, touching one colony is enough, but because sometimes only some of the colonies in a strain produce &bgr;-Iactamasell it is better to touch several colonies. If p-lactamase is produced the colour of the streaked portion of the strip changes from purple to yellow in 5-10 minutes. Non-&bgr;-lactamase-producing strains change the colour of the strip, if at all, to a deeper purple. Known positive and negative controls are applied to each strip. The test is easily read. When dry the colours on test-papers remain for 24 hours. Results
44 strains of H. influenzae, all recently isolated from patients, were tested for &bgr;-Iactamase production by this method and also by two methods in current use in our
laboratory-the chromogenic cephalosporin substrate method’O and a modification of the Kjellander and Myrbäch plate method.8 17 of the 44 strains were included because they had given positive results on earlier routine tests with one of these methods. All 17 gave positive results with all three methods, whereas the remaining 27 strains gave negative results by all three methods. Minimum inhibitory concentrations (M.l.c.) of ampicillin (with carefully standardised inocula and multipoint inoculation on chocolate-agar) were also estimated for 33 of the 44 strains. 21 strains gave MJ.c.s less than 0.6 mg/l, and all these had given negative results in all three tests for p-lactamase production. Of the remaining 13, all of which gave an nt.i.c. of 2.5 mg/tor more, 12 had given positive results in all three &bgr;-lactamase tests. The thirteenth (with an M.l.c. of 20 mgll) had given negative results in all three tests. This strain was therefore clearly an intrinsically resistant strain. Thus there was total agreement between our method and the others in the recognition of ampicillin resistance due to p-lactamase production. Discussion
The test described here is simple and rapid, and reliaidentifies j3-lactamase-producing strains of H. influenzce. It may be performed as soon as there is any visible growth on the culture plates (5-6 hours), and requires only a small number of organisms. This feature could be of value in cases of partially treated meningitis, where there is often only a very scanty growth of the causative organism. The test gives the clinician a more reliable indication of the response he can expect to ampicillin in infections caused by H. influenzae type b than that provided by antibiotic disc sensitivity testing. It is also more rapid. The method may be used for routine surveillance for &bgr;-Iactamase-producing isolates of H. influence. Conventional antibiotic disc sensitivity testing of H. influenzce with ampicillin and chloramphenicol will ensure the detection of the rare ampicillin-resistant, non-p-lactamase producing strains and also those resis-
bly
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., 845. 2. Tomeh, M. O., Starr, S. E., McGowan, J.
Thornsberry, C. Lancet, 1974, ii,
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, 494 9. 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
penicillin-resistant strains of Neisseria gonorrhaeae, kindly supplied by Dr A. Percival, were also tested for p-lactamase production using the method described above. Both strains gave well-defined and posiTwo
tive results within 5 minutes. This suggests that the method may also be used for the routine screening for penicillinase production by gonococci isolated from clinical material.
Methods and Devices A RAPID TEST FOR BETA-LACTAMASE PRODUCTION BY HÆMOPHILUS
INFLUENZÆ D. B. WHELDON MARY P. E. SLACK D. C. TURK
Department of Bacteriology and Regional Public Health Laboratory, Radcliffe Infirmary, Oxford OX2 6HE type b is a common cause of and other acute meningitis life-threatening infections of children which must be treated without delay. In the 1950s chloramphenicol was the treatment of choice. Since the introduction of ampicillin in 1961 many clinicians, especially in the U.S.A., have preferred to use this drug alone, because of fears of chloramphenicol toxicity. In 1974 ampicillin-resistant strains of H. influenzce type b were reported’and are now widely distributed. There is clearly a need for a test which rapidly establishes the true ampicillin sensitivity of strains of H. influenzae type b isolated from clinical material. Ampicillin resistance in H. influenzx is almost always due to the production of p-lactamase, intrinsic resistance being uncommon.33 Several methods for the detection of p-lactamase have been described.4-1O We describe a new rapid acidimetric test for the detection of p-lactamase production by H. influenzae, which uses strips of filter-paper impregnated with penicillin and a pH indicator. These strips may be stored for several months, ready for use when required. The test can be performed as soon as there is visible growth on the primary culture plates. The principle of the method is that any p-lactamase present will hydrolyse benzylpenicillin to form penicilloic acid, with a fall in pH which is demonstrated by a change in the colour of the indicator.
Haemophilus influenzae
Preparation of Test Strips 600 mg of unbuffered benzylpenicillin is dissolved in 4 ml of 0-1% aqueous solution of bromocresol purple. 0.05 ml 0 1 mol/l sodium hydroxide is added. Whatman’s no. 1 filterpapers (9 cm diameter) are immersed in the solution and then immediately dried in a vacuum desiccator with active desiccant. After drying, the paper is cut into strips approximately 0.6cmxl-2cm. The strips are stored at 4°C with silica gel and will retain their activity for several months.
Test One drop of distilled water is placed on a clean microscope slide and covered with a test-paper. The paper should be moist but not oversaturated. A bacteriological loop is used to sweep across the surface of a young culture of the isolate and is then streaked across one end of the strip. In older cultures, touching one colony is enough, but because sometimes only some of the colonies in a strain produce &bgr;-Iactamasell it is better to touch several colonies. If p-lactamase is produced the colour of the streaked portion of the strip changes from purple to yellow in 5-10 minutes. Non-&bgr;-lactamase-producing strains change the colour of the strip, if at all, to a deeper purple. Known positive and negative controls are applied to each strip. The test is easily read. When dry the colours on test-papers remain for 24 hours. Results
44 strains of H. influenzae, all recently isolated from patients, were tested for &bgr;-Iactamase production by this method and also by two methods in current use in our
laboratory-the chromogenic cephalosporin substrate method’O and a modification of the Kjellander and Myrbäch plate method.8 17 of the 44 strains were included because they had given positive results on earlier routine tests with one of these methods. All 17 gave positive results with all three methods, whereas the remaining 27 strains gave negative results by all three methods. Minimum inhibitory concentrations (M.l.c.) of ampicillin (with carefully standardised inocula and multipoint inoculation on chocolate-agar) were also estimated for 33 of the 44 strains. 21 strains gave MJ.c.s less than 0.6 mg/l, and all these had given negative results in all three tests for p-lactamase production. Of the remaining 13, all of which gave an nt.i.c. of 2.5 mg/tor more, 12 had given positive results in all three &bgr;-lactamase tests. The thirteenth (with an M.l.c. of 20 mgll) had given negative results in all three tests. This strain was therefore clearly an intrinsically resistant strain. Thus there was total agreement between our method and the others in the recognition of ampicillin resistance due to p-lactamase production. Discussion
The test described here is simple and rapid, and reliaidentifies j3-lactamase-producing strains of H. influenzce. It may be performed as soon as there is any visible growth on the culture plates (5-6 hours), and requires only a small number of organisms. This feature could be of value in cases of partially treated meningitis, where there is often only a very scanty growth of the causative organism. The test gives the clinician a more reliable indication of the response he can expect to ampicillin in infections caused by H. influenzae type b than that provided by antibiotic disc sensitivity testing. It is also more rapid. The method may be used for routine surveillance for &bgr;-Iactamase-producing isolates of H. influence. Conventional antibiotic disc sensitivity testing of H. influenzce with ampicillin and chloramphenicol will ensure the detection of the rare ampicillin-resistant, non-p-lactamase producing strains and also those resis-
bly
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., 845. 2. Tomeh, M. O., Starr, S. E., McGowan, J.
Thornsberry, C. Lancet, 1974, ii,
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, 494 9. 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
penicillin-resistant strains of Neisseria gonorrhaeae, kindly supplied by Dr A. Percival, were also tested for p-lactamase production using the method described above. Both strains gave well-defined and posiTwo
tive results within 5 minutes. This suggests that the method may also be used for the routine screening for penicillinase production by gonococci isolated from clinical material.
