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. 1997 Feb;41(2):374–378. doi: 10.1128/aac.41.2.374

Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases.

M M Caniça 1, M Barthélémy 1, L Gilly 1, R Labia 1, R Krishnamoorthy 1, G Paul 1
PMCID: PMC163716  PMID: 9021194

Abstract

IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors. The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem. The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates. This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins. The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates. The resistance of E. coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.

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Selected References

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  1. Ambler R. P., Coulson A. F., Frère J. M., Ghuysen J. M., Joris B., Forsman M., Levesque R. C., Tiraby G., Waley S. G. A standard numbering scheme for the class A beta-lactamases. Biochem J. 1991 May 15;276(Pt 1):269–270. doi: 10.1042/bj2760269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Belaaouaj A., Lapoumeroulie C., Caniça M. M., Vedel G., Névot P., Krishnamoorthy R., Paul G. Nucleotide sequences of the genes coding for the TEM-like beta-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiol Lett. 1994 Jul 1;120(1-2):75–80. doi: 10.1111/j.1574-6968.1994.tb07010.x. [DOI] [PubMed] [Google Scholar]
  3. Blazquez J., Baquero M. R., Canton R., Alos I., Baquero F. Characterization of a new TEM-type beta-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli. Antimicrob Agents Chemother. 1993 Oct;37(10):2059–2063. doi: 10.1128/aac.37.10.2059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bret L., Chanal C., Sirot D., Labia R., Sirot J. Characterization of an inhibitor-resistant enzyme IRT-2 derived from TEM-2 beta-lactamase produced by Proteus mirabilis strains. J Antimicrob Chemother. 1996 Aug;38(2):183–191. doi: 10.1093/jac/38.2.183. [DOI] [PubMed] [Google Scholar]
  5. Brun T., Péduzzi J., Caniça M. M., Paul G., Névot P., Barthélémy M., Labia R. Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors. FEMS Microbiol Lett. 1994 Jul 1;120(1-2):111–117. doi: 10.1111/j.1574-6968.1994.tb07016.x. [DOI] [PubMed] [Google Scholar]
  6. Bush K., Jacoby G. A., Medeiros A. A. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother. 1995 Jun;39(6):1211–1233. doi: 10.1128/aac.39.6.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bémer-Melchior P., Gilly L., Jugroot-Klotz K., Brun T., Névot P., Paul G. Etude de la résistance à l'amoxicilline et à l'association amoxicilline-acide clavulanique dans 231 souches cliniques d'Escherichia coli isolées en 1992 à l'hôpital cochin. Pathol Biol (Paris) 1995 Nov;43(9):760–765. [PubMed] [Google Scholar]
  8. Chapon C. Expression of malT, the regulator gene of the maltose region in Escherichia coli, is limited both at transcription and translation. EMBO J. 1982;1(3):369–374. doi: 10.1002/j.1460-2075.1982.tb01176.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chaïbi E. B., Farzaneh S., Péduzzi J., Barthélémy M., Labia R. An additional ionic bond suggested by molecular modelling of TEM-2 might induce a slight discrepancy between catalytic properties of TEM-1 and TEM-2 beta-lactamases. FEMS Microbiol Lett. 1996 Oct 1;143(2-3):121–125. doi: 10.1111/j.1574-6968.1996.tb08470.x. [DOI] [PubMed] [Google Scholar]
  10. Chen S. T., Clowes R. C. Variations between the nucleotide sequences of Tn1, Tn2, and Tn3 and expression of beta-lactamase in Pseudomonas aeruginosa and Escherichia coli. J Bacteriol. 1987 Feb;169(2):913–916. doi: 10.1128/jb.169.2.913-916.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Delaire M., Labia R., Samama J. P., Masson J. M. Site-directed mutagenesis at the active site of Escherichia coli TEM-1 beta-lactamase. Suicide inhibitor-resistant mutants reveal the role of arginine 244 and methionine 69 in catalysis. J Biol Chem. 1992 Oct 15;267(29):20600–20606. [PubMed] [Google Scholar]
  12. Eurocancer 1993. Paris, France, 27-29 avril 1993. Pathol Biol (Paris) 1994 Jan;42(1):1–124. [PubMed] [Google Scholar]
  13. Fonzé E., Charlier P., To'th Y., Vermeire M., Raquet X., Dubus A., Frère J. M. TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant. Acta Crystallogr D Biol Crystallogr. 1995 Sep 1;51(Pt 5):682–694. doi: 10.1107/S0907444994014496. [DOI] [PubMed] [Google Scholar]
  14. Goussard S., Courvalin P. Sequence of the genes blaT-1B and blaT-2. Gene. 1991 Jun 15;102(1):71–73. doi: 10.1016/0378-1119(91)90540-r. [DOI] [PubMed] [Google Scholar]
  15. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henquell C., Chanal C., Sirot D., Labia R., Sirot J. Molecular characterization of nine different types of mutants among 107 inhibitor-resistant TEM beta-lactamases from clinical isolates of Escherichia coli. Antimicrob Agents Chemother. 1995 Feb;39(2):427–430. doi: 10.1128/aac.39.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henquell C., Sirot D., Chanal C., De Champs C., Chatron P., Lafeuille B., Texier P., Sirot J., Cluzel R. Frequency of inhibitor-resistant TEM beta-lactamases in Escherichia coli isolates from urinary tract infections in France. J Antimicrob Chemother. 1994 Nov;34(5):707–714. doi: 10.1093/jac/34.5.707. [DOI] [PubMed] [Google Scholar]
  18. Hibbert-Rogers L. C., Heritage J., Todd N., Hawkey P. M. Convergent evolution of TEM-26, a beta-lactamase with extended-spectrum activity. J Antimicrob Chemother. 1994 Apr;33(4):707–720. doi: 10.1093/jac/33.4.707. [DOI] [PubMed] [Google Scholar]
  19. Knox J. R. Extended-spectrum and inhibitor-resistant TEM-type beta-lactamases: mutations, specificity, and three-dimensional structure. Antimicrob Agents Chemother. 1995 Dec;39(12):2593–2601. doi: 10.1128/aac.39.12.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Labia R., Andrillon J., Le Goffic F. Computerized microacidimetric determination of beta lactamase Michaelis-Menten constants. FEBS Lett. 1973 Jun 15;33(1):42–44. doi: 10.1016/0014-5793(73)80154-1. [DOI] [PubMed] [Google Scholar]
  21. Labia R., Barthélémy M. L'enzymogramme des beta-lactamases: adaptation en cel de la méthode iodométrique. Ann Microbiol (Paris) 1979 Oct;130B(3):295–304. [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lemozy J., Sirot D., Chanal C., Huc C., Labia R., Dabernat H., Sirot J. First characterization of inhibitor-resistant TEM (IRT) beta-lactamases in Klebsiella pneumoniae strains. Antimicrob Agents Chemother. 1995 Nov;39(11):2580–2582. doi: 10.1128/aac.39.11.2580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Matthew M., Hedges R. W. Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility. J Bacteriol. 1976 Feb;125(2):713–718. doi: 10.1128/jb.125.2.713-718.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Oliphant A. R., Struhl K. An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9094–9098. doi: 10.1073/pnas.86.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reguera J. A., Baquero F., Pérez-Díaz J. C., Martínez J. L. Factors determining resistance to beta-lactam combined with beta-lactamase inhibitors in Escherichia coli. J Antimicrob Chemother. 1991 May;27(5):569–575. doi: 10.1093/jac/27.5.569. [DOI] [PubMed] [Google Scholar]
  28. Saves I., Burlet-Schiltz O., Swarén P., Lefèvre F., Masson J. M., Promé J. C., Samama J. P. The asparagine to aspartic acid substitution at position 276 of TEM-35 and TEM-36 is involved in the beta-lactamase resistance to clavulanic acid. J Biol Chem. 1995 Aug 4;270(31):18240–18245. doi: 10.1074/jbc.270.31.18240. [DOI] [PubMed] [Google Scholar]
  29. Stapleton P., Wu P. J., King A., Shannon K., French G., Phillips I. Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli. Antimicrob Agents Chemother. 1995 Nov;39(11):2478–2483. doi: 10.1128/aac.39.11.2478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Thomson C. J., Amyes S. G. TRC-1: emergence of a clavulanic acid-resistant TEM beta-lactamase in a clinical strain. FEMS Microbiol Lett. 1992 Mar 1;70(2):113–117. doi: 10.1016/0378-1097(92)90669-f. [DOI] [PubMed] [Google Scholar]
  32. Vedel G., Belaaouaj A., Gilly L., Labia R., Philippon A., Névot P., Paul G. Clinical isolates of Escherichia coli producing TRI beta-lactamases: novel TEM-enzymes conferring resistance to beta-lactamase inhibitors. J Antimicrob Chemother. 1992 Oct;30(4):449–462. doi: 10.1093/jac/30.4.449. [DOI] [PubMed] [Google Scholar]
  33. Zhou X. Y., Bordon F., Sirot D., Kitzis M. D., Gutmann L. Emergence of clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 beta-lactamase conferring resistance to beta-lactamase inhibitors. Antimicrob Agents Chemother. 1994 May;38(5):1085–1089. doi: 10.1128/aac.38.5.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]

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