ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1977, Copyright 0 1977 American Society for Microbiology
p.
1087-1088
Vol. 11, No. 6 Printed in U.S.A.
Rapid Penicillinase Paper Strip Test for Detection of BetaLactamase-Producing Haemophilus influenzae and Neisseria gonorrhoeae JAMES H. JORGENSEN,* JEAN C. LEE, AND GARY A. ALEXANDER Departments ofPathology* and Microbiology, The University of Texas Health Science Center, and the Bexar County Hospitals, San Antonio, Texas 78284 Received for publication 18 February 1977
A modified 1-min iodometric paper strip test for penicillinase activity was developed for detection of beta-lactamase-producing isolates of Haemophilus influenzae and Neisseria gonorrhoeae. The test is simple to perform and uses reagent-impregnated strips that may be stored for 1 year or more prior to use.
The emergence of ampicillin resistance in Haemophilus influenzae has caused considerable interest in rapid, reliable techniques for recognition of resistant isolates. Several new media formulations and procedures have been described for performance of reliable antimicrobial drug susceptibility tests (3, 4, 5, 9). However, all of these have been modifications of conventional susceptibility test methods that require an additional 18- to 24-h period for completion after the organism is isolated on primary growth media. More recently, penicillinase-producing strains of Neisseria gonorrhoeae have been isolated from at least 11 different countries, including the United States (Morbidity Mortality Weekly Rep. vol. 26, no. 5, p. 29, 4 February 1977). Like the ampicillin-resistant Haemophilus isolates, penicillin-resistant strains of gonococci may occur sporadically in widely separated geographic areas in the future. Several rapid penicillinase tests have recently been described which allow recognition of beta-lactamase-producing (and thus penicillin- and ampicillin-resistant) bacteria as soon as growth is apparent on initial media (1, 2, 7, 8). The present report describes an even more rapid, simplified procedure for detection of penicillinase activity in H. influenzae and N. gonorrhoeae that is based on a method originally described by Shaefler (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, M209, p. 114). Strips of Whatman no. 3 filter paper (approximately 5 by 1 cm) are immersed in a solution of 0.2% soluble starch (BBL) and 1% penicillin (potassium penicillin G for injection). The strips are then allowed to dry at room temperature for approximately 2 h on a perforated metal rack. Dried strips are then stored at
-20°C, which ensures their stability for at least 1 year. To perform the test, a strip is removed from the freezer and placed in a disposable plastic petri dish. The strip is thoroughly moistened with either Gram or Lugol iodine, producing a deep purple color, and excess liquid is then poured off. Approximately 10 colonies of the primary growth of the test organism on chocolate agar or Thayer-Martin medium are then applied to the center of the moistened strip by rubbing it with an inoculating loop in a circular fashion, describing a circle approximately 5 mm in diameter. For purposes of quality control, stock strains of H. influenzae or N. gonorrhoeae showing both ampicillin (or penicillin) resistance and susceptibility are likewise immediately applied to either end of the paper strip. If penicillinase is produced, the deep purple color of the strip becomes white within 1 min in the area where the organisms were applied. If penicillinase is not produced, the strip remains purple or becomes slightly yellow at the immediate site of inoculation. A completed test is considered valid only if the resistant control produces a large white zone, and the susceptible control strain does not. The mechanism of the test involves a preliminary positive starch test as evidenced by the strip turning purple when iodine solution is added. The test area of the strip becomes white if penicilloic acid produced by the action of penicillinase converts the iodine to idoide, which is then no longer available to form the
purple starch-iodine complex. Parallel agar dilution ampicillin susceptibility tests (3) and penicillinase tests performed on a group of 100 isolates (68 susceptible, 32 resistant) of H. influenzae showed agreement be-
1087
1088 NOTES
ANTIMICROB. AGENTs CHEMOTHER.
tween the two procedures in 99 of 100 instances. rior, to previously described methods with reOne strain had an ampicillin minimal inhibi- spect to technical simplicity, time required, and tory concentration (MIC) of 8 ,g/ml, but con- stability of reagents. sistently gave negative penicillinase tests by CITED the present method and two previously de- 1. Catlin, B. W. LITERATURE 1975. lodometric detection of Haemophiscribed techniques (1, 7). This strain would lus influenzae beta-lactamase: rapid presumptive test seem to be unique in prossessing a resistance for ampicillin resistance. Antimicrob. Agents. Chemother. 7:265-270. mechanism other than penicillinase produc2. Escamilla, J. 1976. Susceptibility of Haemophilus inflution, and it bears further investigation. enzae to ampicillin as determined by use of a modiTwenty-seven strains of N. gonorrhoeae were fied, one-minute beta-lactamase test. Antimicrob. examined for penicillinase production and susAgents. Chemother. 9:196-198. ceptibility to penicillin by an agar dilution pro- 3. Jorgensen, J. H., and P. M. Jones. 1975. Simplified medium for ampicillin susceptibility testing of Haecedure that uses Mueller-Hinton agar supplemophilus influenzae. Antimicrob. Agents. Chemomented with 1% IsoVitaleX. Seventeen isolates ther. 7:186-190. were penicillinase test negative and had peni- 4. Jorgensen, J. H., and J. C. Lee. 1975. Microdilution technique for antimicrobial susceptibility testing of cillin MICs of c0.5 U/ml, whereas 10 isolates Haemophilus influenzae. Antimicrob. Agents. Chemwith MICs of :2 U/ml yielded positive penicilother. 8:610-611. linase tests. 5. Khan, W., S. Ross, W. Rodriguez, G. Controni, and A. The current policy in our hospital laboratory K. Saz. 1974. Haemophilus influenzae type B resistant to ampicillin. J. Am. Med. Assoc. 229:298-301. is to perform rapid penicillinase tests on all A., B. Van Klingersen, and Desens-Kroon. clinically significant Haemophilus isolates and 6. Manten, 1976. Chloramphenicol resistance in Haemophilus inall isolates of N. gonorrhoeae. These are folfluenzae. Lancet 1:702. lowed by confirmatory conventional suscepti- 7. Scheifele, D. W., V. P. Syriopoulou, A. L. Harding, B. B. Emerson, and A. L. Smith. 1976. Evaluation of a bility testing of H. influenzae with ampicillin rapid B-lactamase test for detecting ampicillin-resistand chloramphenicol (3). This assures that amant strains of Haenophilus influenzae type b. Pediatpicillin-resistant, but non-penicillinase-producrics 58:382-387. described ing, strains, as well as the recently 8. Thornesberry, C., and L. A. Kirven. 1974. Ampicillin resistance in Haenophilus influenzae as determined chloramphenicol-resistant strains of Haemoby a rapid test for beta-lactamase production. Antiphilus, (6) may be detected. Likewise, any penmicrob. Agents. Chemother. 6:653-654. icillinase-producing isolate of N. gonorrhoeae 9. Thornesberry, C., and L. A. Kirven. 1974. Antimicrodilution susis verified by a conventional agar bial susceptibility of Haemophiluw influenzae. Antimicrob. Agents. Chemother. 6:620-624. ceptibility test. The method that we have deM. O., S. E. Starr, J. E. McGowan, Jr., P. M. scribed is a simpler, more rapid modification of 10. Tomeh, Terry, and A. J. Nahmias. 1974. Ampicillin-resistant the iodometric technique for detection of peniHaemophilus influenzae type B infection. J. Am. cillinase activity. We have found it to be supeMed. Assoc. 229:295-297.
p.
1087-1088
Vol. 11, No. 6 Printed in U.S.A.
Rapid Penicillinase Paper Strip Test for Detection of BetaLactamase-Producing Haemophilus influenzae and Neisseria gonorrhoeae JAMES H. JORGENSEN,* JEAN C. LEE, AND GARY A. ALEXANDER Departments ofPathology* and Microbiology, The University of Texas Health Science Center, and the Bexar County Hospitals, San Antonio, Texas 78284 Received for publication 18 February 1977
A modified 1-min iodometric paper strip test for penicillinase activity was developed for detection of beta-lactamase-producing isolates of Haemophilus influenzae and Neisseria gonorrhoeae. The test is simple to perform and uses reagent-impregnated strips that may be stored for 1 year or more prior to use.
The emergence of ampicillin resistance in Haemophilus influenzae has caused considerable interest in rapid, reliable techniques for recognition of resistant isolates. Several new media formulations and procedures have been described for performance of reliable antimicrobial drug susceptibility tests (3, 4, 5, 9). However, all of these have been modifications of conventional susceptibility test methods that require an additional 18- to 24-h period for completion after the organism is isolated on primary growth media. More recently, penicillinase-producing strains of Neisseria gonorrhoeae have been isolated from at least 11 different countries, including the United States (Morbidity Mortality Weekly Rep. vol. 26, no. 5, p. 29, 4 February 1977). Like the ampicillin-resistant Haemophilus isolates, penicillin-resistant strains of gonococci may occur sporadically in widely separated geographic areas in the future. Several rapid penicillinase tests have recently been described which allow recognition of beta-lactamase-producing (and thus penicillin- and ampicillin-resistant) bacteria as soon as growth is apparent on initial media (1, 2, 7, 8). The present report describes an even more rapid, simplified procedure for detection of penicillinase activity in H. influenzae and N. gonorrhoeae that is based on a method originally described by Shaefler (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, M209, p. 114). Strips of Whatman no. 3 filter paper (approximately 5 by 1 cm) are immersed in a solution of 0.2% soluble starch (BBL) and 1% penicillin (potassium penicillin G for injection). The strips are then allowed to dry at room temperature for approximately 2 h on a perforated metal rack. Dried strips are then stored at
-20°C, which ensures their stability for at least 1 year. To perform the test, a strip is removed from the freezer and placed in a disposable plastic petri dish. The strip is thoroughly moistened with either Gram or Lugol iodine, producing a deep purple color, and excess liquid is then poured off. Approximately 10 colonies of the primary growth of the test organism on chocolate agar or Thayer-Martin medium are then applied to the center of the moistened strip by rubbing it with an inoculating loop in a circular fashion, describing a circle approximately 5 mm in diameter. For purposes of quality control, stock strains of H. influenzae or N. gonorrhoeae showing both ampicillin (or penicillin) resistance and susceptibility are likewise immediately applied to either end of the paper strip. If penicillinase is produced, the deep purple color of the strip becomes white within 1 min in the area where the organisms were applied. If penicillinase is not produced, the strip remains purple or becomes slightly yellow at the immediate site of inoculation. A completed test is considered valid only if the resistant control produces a large white zone, and the susceptible control strain does not. The mechanism of the test involves a preliminary positive starch test as evidenced by the strip turning purple when iodine solution is added. The test area of the strip becomes white if penicilloic acid produced by the action of penicillinase converts the iodine to idoide, which is then no longer available to form the
purple starch-iodine complex. Parallel agar dilution ampicillin susceptibility tests (3) and penicillinase tests performed on a group of 100 isolates (68 susceptible, 32 resistant) of H. influenzae showed agreement be-
1087
1088 NOTES
ANTIMICROB. AGENTs CHEMOTHER.
tween the two procedures in 99 of 100 instances. rior, to previously described methods with reOne strain had an ampicillin minimal inhibi- spect to technical simplicity, time required, and tory concentration (MIC) of 8 ,g/ml, but con- stability of reagents. sistently gave negative penicillinase tests by CITED the present method and two previously de- 1. Catlin, B. W. LITERATURE 1975. lodometric detection of Haemophiscribed techniques (1, 7). This strain would lus influenzae beta-lactamase: rapid presumptive test seem to be unique in prossessing a resistance for ampicillin resistance. Antimicrob. Agents. Chemother. 7:265-270. mechanism other than penicillinase produc2. Escamilla, J. 1976. Susceptibility of Haemophilus inflution, and it bears further investigation. enzae to ampicillin as determined by use of a modiTwenty-seven strains of N. gonorrhoeae were fied, one-minute beta-lactamase test. Antimicrob. examined for penicillinase production and susAgents. Chemother. 9:196-198. ceptibility to penicillin by an agar dilution pro- 3. Jorgensen, J. H., and P. M. Jones. 1975. Simplified medium for ampicillin susceptibility testing of Haecedure that uses Mueller-Hinton agar supplemophilus influenzae. Antimicrob. Agents. Chemomented with 1% IsoVitaleX. Seventeen isolates ther. 7:186-190. were penicillinase test negative and had peni- 4. Jorgensen, J. H., and J. C. Lee. 1975. Microdilution technique for antimicrobial susceptibility testing of cillin MICs of c0.5 U/ml, whereas 10 isolates Haemophilus influenzae. Antimicrob. Agents. Chemwith MICs of :2 U/ml yielded positive penicilother. 8:610-611. linase tests. 5. Khan, W., S. Ross, W. Rodriguez, G. Controni, and A. The current policy in our hospital laboratory K. Saz. 1974. Haemophilus influenzae type B resistant to ampicillin. J. Am. Med. Assoc. 229:298-301. is to perform rapid penicillinase tests on all A., B. Van Klingersen, and Desens-Kroon. clinically significant Haemophilus isolates and 6. Manten, 1976. Chloramphenicol resistance in Haemophilus inall isolates of N. gonorrhoeae. These are folfluenzae. Lancet 1:702. lowed by confirmatory conventional suscepti- 7. Scheifele, D. W., V. P. Syriopoulou, A. L. Harding, B. B. Emerson, and A. L. Smith. 1976. Evaluation of a bility testing of H. influenzae with ampicillin rapid B-lactamase test for detecting ampicillin-resistand chloramphenicol (3). This assures that amant strains of Haenophilus influenzae type b. Pediatpicillin-resistant, but non-penicillinase-producrics 58:382-387. described ing, strains, as well as the recently 8. Thornesberry, C., and L. A. Kirven. 1974. Ampicillin resistance in Haenophilus influenzae as determined chloramphenicol-resistant strains of Haemoby a rapid test for beta-lactamase production. Antiphilus, (6) may be detected. Likewise, any penmicrob. Agents. Chemother. 6:653-654. icillinase-producing isolate of N. gonorrhoeae 9. Thornesberry, C., and L. A. Kirven. 1974. Antimicrodilution susis verified by a conventional agar bial susceptibility of Haemophiluw influenzae. Antimicrob. Agents. Chemother. 6:620-624. ceptibility test. The method that we have deM. O., S. E. Starr, J. E. McGowan, Jr., P. M. scribed is a simpler, more rapid modification of 10. Tomeh, Terry, and A. J. Nahmias. 1974. Ampicillin-resistant the iodometric technique for detection of peniHaemophilus influenzae type B infection. J. Am. cillinase activity. We have found it to be supeMed. Assoc. 229:295-297.
