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Antimicrobial Agents and Chemotherapy, July 2003, p. 2236-2241, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2236-2241.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Phenotypic and Molecular Characterization of Tetracycline- and Erythromycin-Resistant Strains of Streptococcus pneumoniae

Maria P. Montanari, Ileana Cochetti, Marina Mingoia, and Pietro E. Varaldo*

Department of Microbiology and Biomedical Sciences, University of Ancona Medical School, 60131 Ancona, Italy

Received 17 December 2002/ Returned for modification 17 March 2003/ Accepted 15 April 2003

Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, ≥8 µg/ml) and erythromycin (MIC, ≥1 µg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.

* Corresponding author. Mailing address: Department of Microbiology and Biomedical Sciences, University of Ancona Medical School, Via Ranieri, Monte d'Ago, 60131 Ancona, Italy. Phone: 39 071 2204694. Fax: 39 071 2204693. E-mail: pe.varaldo{at}unian.it.

Antimicrobial Agents and Chemotherapy, July 2003, p. 2236-2241, Vol. 47, No. 7
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.7.2236-2241.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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