Phenotypic and molecular characterization of tetracycline- and erythromycin-resistant strains of Streptococcus pneumoniae

Abstract

Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, >or=8 microg/ml) and erythromycin (MIC, >or=1 microg/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.

FIG. 1.

Different fingerprinting profiles obtained by digesting the tet(M) amplicons from 64 tet(M)-positive pneumococci with four endonucleases. Lane M, molecular size marker (100-bp ladder). Lane A, undigested tet(M) amplicon. Lanes 1a to 1e, different AciI profiles (AciI₁ to AciI₅). Lanes 2a and 2b, different RsaI profiles (RsaI₁ and RsaI₂). Lane 3, MseI profile. Lane 4, TaqI profile.

FIG. 2.

HRRA patterns of two tet(M)-positive pneumococci with restriction types A and B. Lane M, molecular size marker (100-bp ladder). Lane A, undigested tet(M) amplicon of the strain exhibiting restriction type A; lanes A1 to A4, restriction profiles yielded by endonucleases AciI, RsaI, MseI, and TaqI, respectively. Lane B, undigested tet(M) amplicon of the strain exhibiting restriction type B; lanes B1 to B4, restriction profiles yielded by endonucleases AciI, RsaI, MseI, and TaqI, respectively.