Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1990 Nov;34(11):2184–2192. doi: 10.1128/aac.34.11.2184

Discrimination of extended-spectrum beta-lactamases by a novel nitrocefin competition assay.

G A Papanicolaou 1, A A Medeiros 1
PMCID: PMC172021  PMID: 2073109

Abstract

We describe a nitrocefin competition assay for determining inhibition profiles as a useful adjunct to existing biochemical methods for the discrimination of beta-lactamases. The hydrolysis rate of nitrocefin was measured with a plate photometer as the change in A480 over 45 min in the presence of 17 inhibitors. Fourteen well-established beta-lactamases and 13 extended-spectrum beta-lactamases were tested. Correlations with data from isoelectric focusing and amino acid sequencing suggested that the inhibition profile reflects alterations in the active-site configuration of beta-lactamases. The method was especially useful in measuring the relative affinities of beta-lactamases against poorly hydrolyzed substrates and in screening large numbers of isolates for the detection of new beta-lactamase types.

Full text

PDF
2188

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ambler R. P., Scott G. K. Partial amino acid sequence of penicillinase coded by Escherichia coli plasmid R6K. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3732–3736. doi: 10.1073/pnas.75.8.3732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ambler R. P. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321–331. doi: 10.1098/rstb.1980.0049. [DOI] [PubMed] [Google Scholar]
  3. Barthélémy M., Peduzzi J., Labia R. Complete amino acid sequence of p453-plasmid-mediated PIT-2 beta-lactamase (SHV-1). Biochem J. 1988 Apr 1;251(1):73–79. doi: 10.1042/bj2510073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barthélémy M., Péduzzi J., Ben Yaghlane H., Labia R. Single amino acid substitution between SHV-1 beta-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme. FEBS Lett. 1988 Apr 11;231(1):217–220. doi: 10.1016/0014-5793(88)80734-8. [DOI] [PubMed] [Google Scholar]
  5. Bauernfeind A., Hörl G. Novel R-factor borne beta-lactamase of Escherichia coli confering resistance to cephalosporins. Infection. 1987 Jul-Aug;15(4):257–259. doi: 10.1007/BF01644127. [DOI] [PubMed] [Google Scholar]
  6. Bush K. Characterization of beta-lactamases. Antimicrob Agents Chemother. 1989 Mar;33(3):259–263. doi: 10.1128/aac.33.3.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bush K., Freudenberger J. S., Sykes R. B. Interaction of azthreonam and related monobactams with beta-lactamases from gram-negative bacteria. Antimicrob Agents Chemother. 1982 Sep;22(3):414–420. doi: 10.1128/aac.22.3.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chanal C. M., Sirot D. L., Labia R., Petit A., Morand A., Sirot J. L., Cluzel R. A. Comparative study of a novel plasmid-mediated beta-lactamase, CAZ-2, and the CTX-1 and CAZ-1 enzymes conferring resistance to broad-spectrum cephalosporins. Antimicrob Agents Chemother. 1988 Nov;32(11):1660–1665. doi: 10.1128/aac.32.11.1660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collatz E., Tran Van Nhieu G., Billot-Klein D., Williamson R., Gutmann L. Substitution of serine for arginine in position 162 of TEM-type beta-lactamases extends the substrate profile of mutant enzymes, TEM-7 and TEM-101, to ceftazidime and aztreonam. Gene. 1989 May 30;78(2):349–354. doi: 10.1016/0378-1119(89)90237-0. [DOI] [PubMed] [Google Scholar]
  10. Eliasson I., Kamme C. Characterization of the plasmid-mediated beta-lactamase in Branhamella catarrhalis, with special reference to substrate affinity. J Antimicrob Chemother. 1985 Feb;15(2):139–149. doi: 10.1093/jac/15.2.139. [DOI] [PubMed] [Google Scholar]
  11. Gutmann L., Ferré B., Goldstein F. W., Rizk N., Pinto-Schuster E., Acar J. F., Collatz E. SHV-5, a novel SHV-type beta-lactamase that hydrolyzes broad-spectrum cephalosporins and monobactams. Antimicrob Agents Chemother. 1989 Jun;33(6):951–956. doi: 10.1128/aac.33.6.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hedges R. W., Datta N., Kontomichalou P., Smith J. T. Molecular specificities of R factor-determined beta-lactamases: correlation with plasmid compatibility. J Bacteriol. 1974 Jan;117(1):56–62. doi: 10.1128/jb.117.1.56-62.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jaurin B., Grundström T. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of beta-lactamases of the penicillinase type. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4897–4901. doi: 10.1073/pnas.78.8.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Joris B., Ghuysen J. M., Dive G., Renard A., Dideberg O., Charlier P., Frère J. M., Kelly J. A., Boyington J. C., Moews P. C. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem J. 1988 Mar 1;250(2):313–324. doi: 10.1042/bj2500313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kliebe C., Nies B. A., Meyer J. F., Tolxdorff-Neutzling R. M., Wiedemann B. Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrob Agents Chemother. 1985 Aug;28(2):302–307. doi: 10.1128/aac.28.2.302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Labia R., Guionie M., Barthélémy M. Properties of three carbenicillin-hydrolysing beta-lactamases (CARB) from Pseudomonas aeruginosa: identification of a new enzyme. J Antimicrob Chemother. 1981 Jan;7(1):49–56. doi: 10.1093/jac/7.1.49. [DOI] [PubMed] [Google Scholar]
  17. Labia R., Morand A., Tiwari K., Sirot J., Sirot D., Petit A. Interactions of new plasmid-mediated beta-lactamases with third-generation cephalosporins. Rev Infect Dis. 1988 Jul-Aug;10(4):885–891. doi: 10.1093/clinids/10.4.885. [DOI] [PubMed] [Google Scholar]
  18. Levesque R. C., Medeiros A. A., Jacoby G. A. Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes. Mol Gen Genet. 1987 Feb;206(2):252–258. doi: 10.1007/BF00333581. [DOI] [PubMed] [Google Scholar]
  19. Mathew A., Harris A. M., Marshall M. J., Ross G. W. The use of analytical isoelectric focusing for detection and identification of beta-lactamases. J Gen Microbiol. 1975 May;88(1):169–178. doi: 10.1099/00221287-88-1-169. [DOI] [PubMed] [Google Scholar]
  20. Medeiros A. A. Beta-lactamases. Br Med Bull. 1984 Jan;40(1):18–27. doi: 10.1093/oxfordjournals.bmb.a071942. [DOI] [PubMed] [Google Scholar]
  21. Medeiros A. A., Cohenford M., Jacoby G. A. Five novel plasmid-determined beta-lactamases. Antimicrob Agents Chemother. 1985 May;27(5):715–719. doi: 10.1128/aac.27.5.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nicolas M. H., Jarlier V., Honore N., Philippon A., Cole S. T. Molecular characterization of the gene encoding SHV-3 beta-lactamase responsible for transferable cefotaxime resistance in clinical isolates of Klebsiella pneumoniae. Antimicrob Agents Chemother. 1989 Dec;33(12):2096–2100. doi: 10.1128/aac.33.12.2096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. O'Callaghan C. H., Morris A., Kirby S. M., Shingler A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. doi: 10.1128/aac.1.4.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paul G. C., Gerbaud G., Bure A., Philippon A. M., Pangon B., Courvalin P. TEM-4, a new plasmid-mediated beta-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli. Antimicrob Agents Chemother. 1989 Nov;33(11):1958–1963. doi: 10.1128/aac.33.11.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Philippon A., Labia R., Jacoby G. Extended-spectrum beta-lactamases. Antimicrob Agents Chemother. 1989 Aug;33(8):1131–1136. doi: 10.1128/aac.33.8.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Péduzzi J., Barthélémy M., Tiwari K., Mattioni D., Labia R. Structural features related to hydrolytic activity against ceftazidime of plasmid-mediated SHV-type CAZ-5 beta-lactamase. Antimicrob Agents Chemother. 1989 Dec;33(12):2160–2163. doi: 10.1128/aac.33.12.2160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Quinn J. P., Miyashiro D., Sahm D., Flamm R., Bush K. Novel plasmid-mediated beta-lactamase (TEM-10) conferring selective resistance to ceftazidime and aztreonam in clinical isolates of Klebsiella pneumoniae. Antimicrob Agents Chemother. 1989 Sep;33(9):1451–1456. doi: 10.1128/aac.33.9.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seeberg A. H., Tolxdorff-Neutzling R. M., Wiedemann B. Chromosomal beta-lactamases of Enterobacter cloacae are responsible for resistance to third-generation cephalosporins. Antimicrob Agents Chemother. 1983 Jun;23(6):918–925. doi: 10.1128/aac.23.6.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shlaes D. M., Medeiros A. A., Kron M. A., Currie-McCumber C., Papa E., Vartian C. V. Novel plasmid-mediated beta-lactamase in members of the family Enterobacteriaceae from Ohio. Antimicrob Agents Chemother. 1986 Aug;30(2):220–224. doi: 10.1128/aac.30.2.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sirot D., Sirot J., Labia R., Morand A., Courvalin P., Darfeuille-Michaud A., Perroux R., Cluzel R. Transferable resistance to third-generation cephalosporins in clinical isolates of Klebsiella pneumoniae: identification of CTX-1, a novel beta-lactamase. J Antimicrob Chemother. 1987 Sep;20(3):323–334. doi: 10.1093/jac/20.3.323. [DOI] [PubMed] [Google Scholar]
  31. Sirot J., Labia R., Thabaut A. Klebsiella pneumoniae strains more resistant to ceftazidime than to other third-generation cephalosporins. J Antimicrob Chemother. 1987 Oct;20(4):611–612. doi: 10.1093/jac/20.4.611. [DOI] [PubMed] [Google Scholar]
  32. Sougakoff W., Goussard S., Gerbaud G., Courvalin P. Plasmid-mediated resistance to third-generation cephalosporins caused by point mutations in TEM-type penicillinase genes. Rev Infect Dis. 1988 Jul-Aug;10(4):879–884. doi: 10.1093/clinids/10.4.879. [DOI] [PubMed] [Google Scholar]
  33. Spencer R. C., Wheat P. F., Winstanley T. G., Cox D. M., Plested S. J. Novel beta-lactamase in a clinical isolate of Klebsiella pneumoniae conferring unusual resistance to beta-lactam antibiotics. J Antimicrob Chemother. 1987 Dec;20(6):919–921. doi: 10.1093/jac/20.6.919. [DOI] [PubMed] [Google Scholar]
  34. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vecoli C., Prevost F. E., Ververis J. J., Medeiros A. A., O'Leary G. P., Jr Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases. Antimicrob Agents Chemother. 1983 Aug;24(2):186–189. doi: 10.1128/aac.24.2.186. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES