Genetic elements carrying erm(B) in Streptococcus pyogenes and association with tet(M) tetracycline resistance gene

Abstract

This study was directed at characterizing the genetic elements carrying the methylase gene erm(B), encoding ribosome modification-mediated resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics, in Streptococcus pyogenes. In this species, erm(B) is responsible for MLS resistance in constitutively resistant isolates (cMLS phenotype) and in a subset (iMLS-A) of inducibly resistant isolates. A total of 125 erm(B)-positive strains were investigated, 81 iMLS-A (uniformly tetracycline susceptible) and 44 cMLS (29 tetracycline resistant and 15 tetracycline susceptible). Whereas all tetracycline-resistant isolates carried the tet(M) gene, tet(M) sequences were also detected in most tetracycline-susceptible isolates (81/81 iMLS-A and 7/15 cMLS). In 2 of the 8 tet(M)-negative cMLS isolates, erm(B) was carried by a plasmid-located Tn917-like transposon. erm(B)- and tet(M)-positive isolates were tested by PCR for the presence of genes int (integrase), xis (excisase), and tndX (resolvase), associated with conjugative transposons of the Tn916 family. In mating experiments using representatives of different combinations of phenotypic and genotypic characteristics as donors, erm(B) and tet(M) were consistently cotransferred, suggesting their linkage in individual genetic elements. The linkage was confirmed by pulsed-field gel electrophoresis and hybridization studies, and different elements, variably associated with the different phenotypes/genotypes, were detected and characterized by amplification and sequencing experiments. A previously unreported genetic organization, observed in all iMLS-A and some cMLS isolates, featured an erm(B)-containing DNA insertion into the tet(M) gene of a defective Tn5397, a Tn916-related transposon. This new element was designated Tn1116. Genetic elements not previously described in S. pyogenes also included Tn6002, an unpublished transposon whose complete sequence is available in GenBank, and Tn3872, a composite element resulting from the insertion of the Tn917 transposon into Tn916 [associated with a tet(M) gene expressed in some cMLS isolates and silent in others]. The high frequency of association between a tetracycline-susceptible phenotype and tet(M) genes suggests that transposons of the Tn916 family, so far typically associated solely with a tetracycline-resistant phenotype, may be more widespread in S. pyogenes than currently believed.

FIG. 1.

Schematic representation of Tn1116, a novel Tn916 family transposon resulting from the insertion of erm(B)-containing DNA into the tet(M) gene of a defective Tn5397. The unsequenced region is tentatively represented upstream of the sequenced region. A squared arrow indicates erm(B); a striped arrow indicates tet(M). The distance between erm(B) and tet(M) was 1,090 bp. Thin arrows below erm(B) and tet(M) indicate the positions and directions of the primers used to investigate the linkage between the two resistance genes. The inverted repeat sequence (IR) upstream of the IS1216 transposase was incomplete. The nucleotide sequence of the DNA region between the erm(B) gene and the tndX gene has been deposited under accession no. AM411377.

FIG. 2.

Schematic representation of the linkage between genes erm(B) (squared arrow) and tet(M) (striped arrow) in isolates displaying P/G combination B. The genetic organization was fully consistent with that of Tn6002, a Tn916-related, erm(B)-containing unpublished transposon (formerly called Tn916Erm) whose complete sequence is available in GenBank (accession no. AY898750). ORFs other than tet(M) that are shared by Tn6002 and Tn916 are represented as gray arrows; ORFs other than erm(B) that are not present in Tn916 are represented as black arrows. Black bars above the corresponding ORFs indicate the amplicons (whose sizes are expressed in bp) obtained with the primers whose sequences are reported in Table 1. Thin arrows below erm(B) and tet(M) indicate the positions and directions of the primers used to investigate the linkage between the two resistance genes.

FIG. 3.

Schematic representation of the linkage between genes erm(B) (squared arrow) and tet(M) (striped arrow) in isolates displaying P/G combinations C and D. The genetic organization was consistent with that of Tn3872, a composite element resulting from the insertion of the Tn917 element (ca. 5.3 kb) into Tn916 (ca. 18 kb), except for the lack of PCR evidence of the two genes (orf24 and int) located on the left and right end of the transposon. ORFs of Tn916 other than tet(M) are represented as gray arrows (white arrows with dashed outlines indicate ORFs undetected by PCR). ORFs of Tn917 other than erm(B) are represented as black arrows. Black bars above the corresponding ORFs indicate the amplicons (whose sizes are expressed in bp) obtained with the primers whose sequences are reported in Table 1. Thin arrows below erm(B) and tet(M) indicate the positions and directions of the primers used to investigate the linkage between the two resistance genes. The nucleotide sequence of the DNA region between the tet(M) gene and the erm(B) gene has been deposited under accession no. AM411624.