Abstract
DNA-DNA hybridization and sequencing were performed to determine the molecular basis of resistance to clavulanic acid in 107 inhibitor-resistant TEM (IRT) enzymes produced by Escherichia coli clinical isolates. These beta-lactamases derived from TEM-1 enzyme focused at pI 5.2 (n = 68) or 5.4 (n = 39) and were very poorly inhibited by clavulanic acid compared with TEM-1 enzyme. Results showed that the amino acid sequences of 84 of the 107 enzymes differ from TEM-1 by one or two substitutions previously described: Arg-244-->Ser (IRT-2) in 22 strains, Met-69-->Leu (TEM-33) in 17 strains, Met-69-->Val (TEM-34) in 14 strains, Met-69-->Ile (IRT-3) in 6 strains, Met-69-->Leu associated with Asn-276-->Asp (IRT-4) in 13 strains, and Met-69-->Val associated with Asn-276-->Asp (TEM-36) in 12 strains. A new combination, Met-69-->Ile with Asn-276-->Asp, was found in 20 strains and was called IRT-8. Two IRT enzymes not previously described were characterized. The substitution Met-69-->Val associated with a novel substitution Arg-275-->Leu occurred in one strain. The combination Met-69-->Leu and Asn-276-->Asp was associated with the novel substitution Trp-165-->Arg in two strains. These two novel enzymes were called IRT-9 and IRT-10, respectively. The implication of these novel mutated positions, 165 and 275, in resistance to inactivation by clavulanate was supported by crystallographic data on the TEM-1 enzyme and results of site-directed mutagenesis. Molecular characterization of these mutants showed great diversity among the genes coding for inhibitor-resistant TEM enzymes produced by clinical E. coli isolates.
Full Text
The Full Text of this article is available as a PDF (182.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- 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]
- 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]
- Chanal C., Poupart M. C., Sirot D., Labia R., Sirot J., Cluzel R. Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes. Antimicrob Agents Chemother. 1992 Sep;36(9):1817–1820. doi: 10.1128/aac.36.9.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Jacoby G. A., Medeiros A. A. More extended-spectrum beta-lactamases. Antimicrob Agents Chemother. 1991 Sep;35(9):1697–1704. doi: 10.1128/aac.35.9.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jelsch C., Lenfant F., Masson J. M., Samama J. P. Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution. FEBS Lett. 1992 Mar 9;299(2):135–142. doi: 10.1016/0014-5793(92)80232-6. [DOI] [PubMed] [Google Scholar]
- Mabilat C., Courvalin P. Development of "oligotyping" for characterization and molecular epidemiology of TEM beta-lactamases in members of the family Enterobacteriaceae. Antimicrob Agents Chemother. 1990 Nov;34(11):2210–2216. doi: 10.1128/aac.34.11.2210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sirot D., Chanal C., Henquell C., Labia R., Sirot J., Cluzel R. Clinical isolates of Escherichia coli producing multiple TEM mutants resistant to beta-lactamase inhibitors. J Antimicrob Chemother. 1994 Jun;33(6):1117–1126. doi: 10.1093/jac/33.6.1117. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]