Role of penicillin-binding protein 5 C-terminal amino acid substitutions in conferring ampicillin resistance in Norwegian clinical strains of Enterococcus faecium
R. JUREEN,
S. C. MOHN,
S. HARTHUG, L. HAARR,
N. LANGELAND,
Corresponding Author
R. JUREEN
Institute of Medicine,
Roland Jureen, Institute of Medicine, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. e-mail: [email protected]Search for more papers by this authorS. C. MOHN
Institute of Medicine,
Center for Medical Genetics and Molecular Medicine, Department of
Search for more papers by this authorL. HAARR
Microbiology and Immunology, University of Bergen, Departments of
Search for more papers by this authorN. LANGELAND
Institute of Medicine,
Medicine, Haukeland University Hospital, Bergen, Norway
Search for more papers by this authorR. JUREEN,
S. C. MOHN,
S. HARTHUG, L. HAARR,
N. LANGELAND,
Corresponding Author
R. JUREEN
Institute of Medicine,
Roland Jureen, Institute of Medicine, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. e-mail: [email protected]Search for more papers by this authorS. C. MOHN
Institute of Medicine,
Center for Medical Genetics and Molecular Medicine, Department of
Search for more papers by this authorL. HAARR
Microbiology and Immunology, University of Bergen, Departments of
Search for more papers by this authorN. LANGELAND
Institute of Medicine,
Medicine, Haukeland University Hospital, Bergen, Norway
Search for more papers by this authorAbstract
The importance of amino acid sequence differences in the C-terminal part and levels of mRNA expression of penicillin-binding protein 5 (PBP5) for ampicillin resistance in Enterococcus faecium was investigated. Seventeen isolates from Norwegian hospitalized patients (ampicillin MIC 0.064->256 mg/L) with different C-terminal pbp5 DNA sequences encoding 11 different amino acid sequences were analyzed with a 14C-radiolabeled penicillin- binding assay to PBP5 and with real-time PCR quantification of pbp5 mRNA expression. Using multiple logistic regression analysis the amino acid substitution Met 485 was linked to ampicillin MIC and levels of 14C-radiolabeled penicillin bound to PBP5; however, there were isolates with identical PBP5 alleles and different ampicillin MICs. There was no relation between the quantity of pbp5 mRNA transcripts and ampicillin resistance. The results cannot explain ampicillin resistance in Norwegian clinical strains of E. faecium and indicate that other factors besides the properties of the C-terminal part of PBP5 are most likely involved.
REFERENCES
- 1 Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev 1990; 3: 46–65.
- 2 Morrison AJ Jr, Wenzel RP. Nosocomial urinary tract infections due to enterococcus. Ten years' experience at a university hospital. Arch Intern Med 1986; 146: 1549–51.
- 3 Patterson JE, Sweeney AH, Simms M, Carley N, Mangi R, Sabetta J, et al. An analysis of 110 serious enterococcal infections. Epidemiology, antibiotic susceptibility, and outcome. Medicine (Baltimore) 1995; 74: 191–200.
- 4 Maki DG, Agger WA. Enterococcal bacteremia: clinical features, the risk of endocarditis, and management. Medicine (Baltimore) 1988; 67: 248–69.
- 5 Huycke MM, Sahm DF, Gilmore MS. Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg Infect Dis 1998; 4: 239–49.
- 6 Torell E, Cars O, Hambraeus A. Ampicillin-resistant enterococci in a Swedish university hospital: nosocomial spread and risk factors for infection. Scand J Infect Dis 2001; 33: 182–7.
- 7 Woodford N, Johnson AP, Morrison D, Speller DC. Current perspectives on glycopeptide resistance. Clin Microbiol Rev 1995; 8: 585–615.
- 8 Bonten MJ, Willems R, Weinstein RA. Vancomycin-resistant enterococci: why are they here, and where do they come from? Lancet Infect Dis 2001; 1: 314–25.
- 9 Simonsen GS, Smabrekke L, Monnet DL, Sorensen TL, Moller JK, Kristinsson KG, et al. Prevalence of resistance to ampicillin, gentamicin and vancomycin in Enterococcus faecalis and Enterococcus faecium isolates from clinical specimens and use of antimicrobials in five Nordic hospitals. J Antimicrob Chemother 2003; 51: 323–31.
- 10 Ghuysen JM. Molecular structures of penicillin-binding proteins and beta-lactamases. Trends Microbiol 1994; 2: 372–80.
- 11 Zorzi W, Zhou XY, Dardenne O, Lamotte J, Raze D, Pierre J, et al. Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium. J Bacteriol 1996; 178: 4948–57.
- 12 Sifaoui F, Arthur M, Rice L, Gutmann L. Role of penicillin-binding protein 5 in expression of ampicillin resistance and peptidoglycan structure in Enterococcus faecium. Antimicrob Agents Chemother 2001; 45: 2594–7.
- 13 Ligozzi M, Pittaluga F, Fontana R. Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 1996; 40: 354–7.
- 14 Signoretto C, Canepari P. Paradoxical effect of inserting, in Enterococcus faecalis penicillin- binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium. Arch Microbiol 2000; 173: 213–9.
- 15 Rybkine T, Mainardi JL, Sougakoff W, Collatz E, Gutmann L. Penicillin-binding protein 5 sequence alterations in clinical isolates of Enterococcus faecium with different levels of beta-lactam resistance. J Infect Dis 1998; 178: 159–63.
- 16 Harthug S, Jureen R, Mohn SC, Digranes A, Simonsen GS, Sundsfjord A, et al. The prevalence of faecal carriage of ampicillin-resistant and high-level gentamicin-resistant enterococci among inpatients at 10 major Norwegian hospitals. J Hosp Infect 2002; 50: 145–54.
- 17 Fontana R, Aldegheri M, Ligozzi M, Lopez H, Sucari A, Satta G. Overproduction of a low-affinity penicillin-binding protein and high- level ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 1994; 38: 1980–3.
- 18 Rice LB, Carias LL, Hutton-Thomas R, Sifaoui F, Gutmann L, Rudin SD. Penicillin-binding protein 5 and expression of ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 2001; 45: 1480–6.
- 19 Harthug S, Eide GE, Langeland N. Nosocomial outbreak of ampicillin resistant Enterococcus faecium: risk factors for infection and fatal outcome. J Hosp Infect 2000; 45: 135–44.
- 20 Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995; 33: 24–7.
- 21 Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995; 33: 1434.
- 22 Jureen R, Top J, Mohn SC, Harthug S, Langeland N, Willems RJ. Molecular characterization of ampicillin-resistant Enterococcus faecium isolates from hospitalized patients in Norway. J Clin Microbiol 2003; 41: 2330–6.
- 23 NCCLS. National Commitee for Laboratory Standards (NCCLS). Performance standards for antimicrobial susceptibility testing (M100-S11). Wayne, PA: NCCLS, 2001.
- 24 Murray BE, Singh KV, Heath JD, Sharma BR, Weinstock GM. Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. J Clin Microbiol 1990; 28: 2059–63.
- 25 Dahl KH, Simonsen GS, Olsvik O, Sundsfjord A. Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci. Antimicrob Agents Chemother 1999; 43: 1105–10.
- 26 Schutz E, Von Ahsen N. Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches. Biotechniques 1999; 27: 1218–22, 1224.
- 27 Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986–94.
- 28 Mohn SC, Ulvik A, Jureen R, Willems RJ, Top J, Leavis H, et al. Duplex real-time PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium. Antimicrob Agents Chemother 2004; 48: 556–560.
- 29 Sauvage E, Kerff F, Fonze E, Herman R, Schoot B, Marquette JP, et al. The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin. Cell Mol Life Sci 2002; 59: 1223–32.
- 30 Snyder L, Champness W. Molecular Genetics of Bacteria. Washington, D.C.: ASM Press 1997.
- 31 Cortazzo P, Cervenansky C, Marin M, Reiss C, Ehrlich R, Deana A. Silent mutations affect in vivo protein folding in Escherichia coli. Biochem Biophys Res Commun 2002; 293: 537–41.
- 32 Mollerach ME, Partoune P, Coyette J, Ghuysen JM. Importance of the E-46-D-160 polypeptide segment of the non-penicillin- binding module for the folding of the low-affinity, multimodular class B penicillin-binding protein 5 of Enterococus hirae. J Bacteriol 1996; 178: 1774–5.
