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Antimicrobial Agents and Chemotherapy, January 2005, p. 32-39, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.32-39.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Differences in the Enterococcus faecalis lsa Locus That Influence Susceptibility to Quinupristin-Dalfopristin and Clindamycin

Kavindra V. Singh^1,2 and Barbara E. Murray^1,2,3*

Center for the Study of Emerging and Reemerging Pathogens,¹ Department of Internal Medicine, Division of Infectious Diseases,² Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, Houston, Texas³

Received 2 June 2004/ Returned for modification 19 July 2004/ Accepted 18 September 2004

We have previously shown that the Enterococcus faecalis lsa gene, encoding the putative ABC protein Lsa, influences resistance to quinupristin-dalfopristin (Q-D) and clindamycin (CLI). We have now found that, while cloned lsa from E. faecalis strain V583 (lsa_V) fully restored resistance to Q-D, CLI, and dalfopristin (DAL) lost by the OG1 lsa disruption mutant TX5332 and also caused increased MICs for Lactococcus lactis LM2301, cloned lsa from OG1 (lsa_OG) did not cause any increase in MICs for either species. Sequencing of ca. 2 kb of these two lsa alleles found differences between lsa_OG and lsa_V in the upstream region as well as in the 5' and 3' halves of the lsa gene. To investigate the reason for the phenotypic differences expressed by the two cloned loci, 5' half plus 3' half hybrid constructs were created. When introduced into both TX5332 and L. lactis, cloned lsa_V5'OG3' conferred increases in MICs of Q-D, CLI, and DAL similar to those of cloned lsa_V while cloned lsa_OG5'V3' showed a moderate increase in MICs relative to those of lsa_OG, indicating that both halves of the locus can influence resistance expression. After site-directed mutagenesis of the cloned lsa alleles at positions –131 and –133 (relative to the putative Lsa start codon ATG), which converted two A's of lsa_V to the G and T of lsa_OG and vice versa, MIC testing showed that mutagenized lsa_OG (lsa_OG-M) was strongly influenced by these changes in terms of conferring increased MICs of Q-D, CLI, and DAL relative to lsa_OG while the phenotype of mutagenized lsa_V (lsa_V-M) was less influenced, with moderately decreased MICs, primarily to CLI, relative to lsa_V. In conclusion, this study found that changes in different regions of the E. faecalis lsa locus influence the ability of cloned lsa to confer resistance to Q-D, CLI, and DAL.

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* Corresponding author. Mailing address: Center for the Study of Emerging and Reemerging Pathogens, University of Texas Medical School—Houston, 6431 Fannin, 2.112MSB, Houston TX 77030. Phone: (713) 500-6745. Fax: (713) 500-5495. E-mail: bem.asst{at}uth.tmc.edu.

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Antimicrobial Agents and Chemotherapy, January 2005, p. 32-39, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.32-39.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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