Tn3701, carried by Streptococcus pyogenes A454, is the first chromosomal composite element to be described; it contains in its central region Tn3703, a transposon similar to Tn916. A comparison by DNA-DNA hybridization of Tn3701 with omega(cat-tet) and Tn3951, carried by Streptococcus pneumoniae BM6001 and by Streptococcus agalactiae B109, respectively, revealed that the two latter structures are also Tn3701-like composite elements. The DNAs of 27 other antibiotic-resistant group A, B, C, and G streptococci and of S. pneumoniae BM4200 showed sequence homologies to Tn3701 (14 strains, including BM4200), to the regions of Tn3701 outside of Tn3703 (5 strains), and to Tn916 alone (8 strains). The DNAs of five strains did not detectably hybridize with any probe. The tetM gene was identified in most chromosomal genetic elements coding for tetracycline-minocycline resistance. Since Tn3701-like elements are widely disseminated among antibiotic-resistant streptococci (47% of the 34 strains studied), we propose that Tn3701 be considered the prototype of chromosomal composite elements.

The plasmid-free Streptococcus pyogenes A454 contains a conjugative element, Tn37O1, encoding resistance to erythromycin (Emr), tetracycline (Tcr), and minocycline (Mn'). We have mapped a 50-kilobase (kb) chromosomal region of A454 corresponding to the internal part of Tn34701. Tn3701 includes a 19.7-kb structure, designated Tn3703, on which the Emr Tcr Mnr determinants were localized. Tn3703 was very similar in structure to Tn916. Translocation of the Emr Tcr Mnr markers from A454 onto pIP964, an Enterococcus faecalis hemolysin plasmid, yielded different pIP964 derivatives. When the inserts of four of these derivatives were aligned with the 50-kb region of Tn3701, three of them were found to result from the transposition of Tn3703 and one resulted from the insertion of a 44.0-kb portion of Tn3701, including Tn3703. Tn3701 inserted, apparently without changing its structure, in the chromosomes of various streptococcal transconjugants, as well as in one of the 12 E. faecalis transconjugants studied. Tn3703 inserted at different chromosomal sites in four E. faecalis transconjugants, and one copy of Tn3701 plus an additional copy of Tn3703 were detected in the chromosomes of seven transconjugants.

An approach based on PCR has been developed to identify new members of the tet gene family in streptococci resistant to tetracycline and minocycline. Degenerate primers, corresponding to portions of the conserved domains of the proteins Tet(M), Tet(O), TeTB(P), Tet(Q), and Tet(S), all specifying the tetracycline-minocycline resistance phenotype, were used to selectively amplify DNA fragments within the coding sequences. Nine streptococcal strains which do not carry the genes tet(M), tet(O), tetB(P), tet(Q), or tet(S) were investigated. Four of them gave no detectable PCR products. The five remaining strains each yielded a PCR product of 1.1 kbp. DNA hybridization experiments showed that these putative Tet determinants fell into four new hybridization classes, of which one, Tet T, was further analyzed. The gene tet(T) was isolated from Streptococcus pyogenes A498, and the nucleotide sequence that was necessary and sufficient for the expression of tetracycline resistance in Escherichia coli was determined. The deduced Tet(T) protein consists of 651 amino acids. The protein most closely related to Tet(T) was Tet(Q), which has 49% identical amino acid residues. A phylogenetic analysis revealed that Tet T represents a novel branching order among the Tet determinants so far described.

Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the insertion sequence IS1182. This transposon carries two aminoglycosides resistance genes, aphA-3 and aadE, an altered gene similar to sat4 from Campylobacter coli BE/G4, and three open reading frames of unknown functions.

An assay based on the utilization of degenerate primers that enable enzymatic amplification of an internal fragment of cat genes known to be present in gram-positive cocci was developed to identify the genes encoding chloramphenicol resistance in streptococci and enterococci. The functionality of this system was illustrated by the detection of cat genes belonging to four different hydridization classes represented by the staphylococcal genes catpC221, catpC1949 catpSCS79 and the clostridial gene catP, and by the characterization of a new streptococcal cat gene designated catS. A sequence related to the clostridial catQ gene, which was present in one streptococcal strain, was not detected by this assay. These results reveal that these six cat genes account for chromosomal-borne chloramphenicol resistance in 12 group A, B, and G streptococci tested. By contrast, only three of these six cat genes (catp'221, catj%94 and catpsCS7) were detected on the 10 enterococcal plasmids studied here that encode resistance to chloramphenicol.Chloramphenicol resistance (Cmr) in bacteria of clinical importance is generally due to the synthesis of the enzyme chloramphenicol acetyltransferase (CAT) which inactivates the antibiotic by converting it successively to 3-acetyl and 1,3-diacetyl derivatives (31). Comparison of the amino acid sequences of 17 CAT proteins from gram-negative and gram-positive bacteria has revealed a significant degree of homology between the various enzymes, and their phylogenetic relationship has been established (2, 18, 29). Nucleotide sequences are now available for 13 cat genes originating in gram-positive bacteria. These are the gene cat-86 from Bacillus pumilus (10); those carried by the staphylococcal plasmids pC194 (11), pC221 (32), pSCS1 (30), pSCS5 (28), pSCS6 (7), pSCS7 (29), and pUB112 (5) and by the streptococcal plasmid pIP501 (12,35) Utilization of degenerate primers in polymerase chain reaction (PCR) enables the detection of rRNA methylase genes that confer resistance to macrolide-lincosamide-streptogramin B-type (MLS) antibiotics in gram-positive bacteria (1). We have developed a similar assay in order to characterize the cat genes present in the remaining seven plasmidfree streptococci (23) and in the three Enterococcus faecalis plasmids (26) which did not detectably hybridize with catp.221 (or catpIP501) and cat C194* In addition, one E. faecalis and six Enterococcus [aecium plasmids coding for Cmr were included in this study. MATERIALS AND METHODSBacterial strains and culture conditions. The main characteristics of the strains and plasmids used in this study which carried unidentified cat genes are listed in Table 1

The tetracycline (Tet) determinants, which encode resistance either to tetracyclines without minocycline (Tcr)

Eight wild-type strains of Enterococcusfaecalis, resistant to chloramphenicol (Cm'), erythromycin (Emr), tetracycline (Tcr), and minocycline (Mnr), were examined for the genetic basis of their antibiotic resistance. Five of the strains transferred all of their antibiotic resistance markers by conjugation, while the other three strains transferred only Tcr and Mnr. Cmr and Emr determinants were localized by DNA-DNA hybridization experiments, in which the Cmr gene of plasmid pIP501, of group B Streptococcus origin, and the Emr gene of transposon Tn917, of E. faecalis origin, served as probes. A chromosomal location was found for the nonconjugative Cmr and Emr markers of one wild-type strain. In two strains these markers were carried by nonconjugative plasmids, and in the other strains they were carried by plasmids that transferred by conjugation. Plasmids isolated from three transconjugants resistant to tetracycline but susceptible to minocycline bore nucleotide sequences homologous to the tetL gene. Nucleotide sequences homologous to conjugative transposon Tn916, of E. faecalis origin, were detected by hybridization in the tetracycline-minocycline-resistant transconjugants. Three of these transconjugants were plasmid free, while four harbored conjugative cryptic plasmids. Sequences homologous to Tn916 were also found on two conjugative plasmids, one of which appeared to be a conjugative cryptic plasmid that had acquired chromosomal Tcr Mnr markers during transfer.

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