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Antimicrobial Agents and Chemotherapy, September 2001, p. 2594-2597, Vol. 45, No. 9
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.9.2594-2597.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Role of Penicillin-Binding Protein 5 in Expression of Ampicillin Resistance and Peptidoglycan Structure in Enterococcus faecium

Farid Sifaoui,1 Michel Arthur,1 Louis Rice,2 and Laurent Gutmann1,*

L.R.M.A., INSERM E0004, Université Paris VI, 75270 Paris Cedex 06, France,1 and Medical and Research Services, Case Western University, Cleveland, Ohio2

Received 20 February 2001/Returned for modification 25 April 2001/Accepted 6 June 2001

The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired beta -lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 µg/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 µg/ml), D344 (MIC, 24 µg/ml), and H80721 (MIC, 512 µg/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 µg/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.

* Corresponding author. Mailing address: L.R.M.A./E0004, Université Paris VI, 15, rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France. Phone: 33-1-42.34.68.63. Fax: 33-1-43.25.68.12. E-mail: laurent.gutmann{at}ccr.jussieu.fr.

Antimicrobial Agents and Chemotherapy, September 2001, p. 2594-2597, Vol. 45, No. 9
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.9.2594-2597.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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