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Antimicrobial Agents and Chemotherapy, November 2005, p. 4622-4627, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4622-4627.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Amino Acid Mutations Essential to Production of an Altered PBP 2X Conferring High-Level ß-Lactam Resistance in a Clinical Isolate of Streptococcus pneumoniae

Anthony M. Smith1* and Keith P. Klugman1,2

Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, Medical Research Council, and University of the Witwatersrand, Johannesburg, South Africa,1 Department of Global Health, Rollins School of Public Health, and Division of Infectious Diseases, School of Medicine, Emory University, Atlanta, Georgia2

Received 23 May 2005/ Returned for modification 10 July 2005/ Accepted 5 August 2005

Altered penicillin-binding protein 2X (PBP 2X) is a primary ß-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 µg/ml; cefotaxime MIC, 4 µg/ml). Strain R62X/2B/1A/mur (for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R62X/2B/1A/mur. Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R62X/2B/1A/mur. Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 µg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R62X/2B/1A/mur to 0.06 µg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.

* Corresponding author. Mailing address: Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, Private Bag X4, Sandringham 2131, South Africa. Phone: 27-11-5550353. Fax: 27-11-5550437. E-mail: anthony.smith{at}nhls.ac.za.

Antimicrobial Agents and Chemotherapy, November 2005, p. 4622-4627, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4622-4627.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zerfass, I., Hakenbeck, R., Denapaite, D. (2009). An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae: Mutational Analysis of Thr338. Antimicrob. Agents Chemother. 53: 1107-1115 [Abstract] [Full Text]  
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