Diversity of structures carrying the high-level gentamicin resistance gene (aac6-aph2) in Enterococcus faecalis strains isolated in France.

Of 24 high-level gentamicin-resistant clinical isolates of Enterococcus faecalis, 20 carried gentamicin resistance (Gmr) plasmids. The plasmids ranged from 65.0 to 80.

Molecular typing of Enterococcus faecalis strains resistant to high levels of gentamicin and isolated in Romania.

Sixteen Enterococcus faecalis strains resistant to high levels of gentamicin, 15 of which were isolated in the same year in a Romanian hospital, harboured conjugative gentamicin resistance (Gm(r)) plasmids ranging from 55 to 85 kilobases. On the basis of restriction enzyme and DNA-DNA hybridization profiles of these plasmids, as well as of chromosomal SmaI macrorestriction and Tn916 hybridization patterns, clonal relationship was established for seven strains whereas the other strains were considered to be independent. Nine and seven of the Gm(r) plasmids carried Tn4001-like and Tn4001-truncated structures, respectively; the latter structures were truncated in the right-hand flanking extremity of the element.

New tetracycline resistance determinants coding for ribosomal protection in streptococci and nucleotide sequence of tet(T) isolated from Streptococcus pyogenes A498.

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.

Plasmid-borne high-level resistance to gentamicin in Enterococcus hirae, Enterococcus avium, and Enterococcus raffinosus.

Enterococcus hirae, E. avium, and E. raffinosus isolated in Romania, Tunisia, and Portugal harbored plasmids pICC8, pIP1700, and pIP1701, respectively, encoding resistance to high levels of gentamicin (Gmr).

Chromosomal gentamicin resistance transposon Tn3706 in Streptococcus agalactiae B128.

Streptococcus agalactiae B128 is the only highly gentamicin-resistant group B streptococcal (GBS) strain described so far. This strain carries a chromosomal gentamicin resistance transposon, designated Tn3706, which is similar, if not identical, to the Tn4001 and Tn5281 transpons detected in Staphylococcus aureus and Enterococcus faecalis, respectively. Transposition of Tn3706 occurred onto the GBS plasmid pIP501 in two different loci of its 7.

Genetic and molecular studies of a composite chromosomal element (Tn3705) containing a Tn916-modified structure (Tn3704) in Streptococcus anginosus F22.

The plasmid-free Streptococcus anginosus F22 contained a conjugative element, Tn3705, encoding resistance to erythromycin (Emr) and tetracycline-minocycline (Tcr-Mnr). We mapped a chromosomal region (> 52 kb) of F22, corresponding to the internal part of Tn3705. Molecular analysis of Tn3705 revealed it to be a composite structure: it included in its central part a transposon designated Tn3704 (20.

Study of heterogeneity of chloramphenicol acetyltransferase (CAT) genes in streptococci and enterococci by polymerase chain reaction: characterization of a new CAT determinant.

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, catpC194, catpSCS7, 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.

Diversity of chromosomal genetic elements and gene identification in antibiotic-resistant strains of Streptococcus pneumoniae and Streptococcus bovis.

Antibiotic-resistant Streptococcus pneumoniae (26 strains) and Streptococcus bovis (28 strains), devoid of R plasmids, were examined for DNA-DNA homology to Tn916 and Tn3701. Tn916-like structures were found in 17 S. pneumoniae and 21 S.

Nucleotide sequence of the chloramphenicol resistance determinant of the streptococcal plasmid pIP501.

We have sequenced the chloramphenicol resistance determinant (cat) of plasmid pIP501 from Streptococcus agalactiae to investigate its relationship with other cognate cat determinants. Sequence analysis revealed that it exhibits a high degree of similarity with the cat genes of plasmids pC221 and pUB112 from Staphylococcus aureus and pSCS1 from Staphylococcus intermedius. These genes, however, display several differences in their regulatory and coding regions.

Identical genes confer high-level resistance to gentamicin upon Enterococcus faecalis, Enterococcus faecium, and Streptococcus agalactiae.

The structural gene coding for the bifunctional aminoglycoside-modifying 6'-acetyltransferase-2''-phosphotransferase (6'AAC-2''APH) enzyme was specifically amplified by the polymerase chain reaction using template DNA of clinical isolates of enterococci (both Enterococcus faecalis and Enterococcus faecium) and the single high-level gentamicin-resistant Streptococcus agalactiae strain identified thus far. The results of the present study demonstrated that the genes encoding this antibiotic resistance trait are highly homologous in these species. In dot blot hybridization assays using nonradioactively labeled oligonucleotide probes, strains with and without high-level gentamicin resistance could be discerned unequivocally.

Identification of new sex pheromone plasmids in Enterococcus faecalis.

We describe the identification of the following new sex pheromone plasmids in Enterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.

Natural occurrence of structures in oral streptococci and enterococci with DNA homology to Tn916.

Seventeen oral streptococci and 18 enterococci were tested for the presence of DNA sequences homologous to the conjugative transposon Tn916 encoding tetracycline resistance. All the strains were resistant to tetracyclines, including minocycline, and most of them were resistant to other antibiotics. Tn916-like structures, identified by hybridization of HincII-digested DNA, were found on the chromosomes of 11 oral streptococci and four enterococci and on two plasmids, pIP1549 and pIP1440, one harbored by an Enterococcus hirae strain and the other harbored by an Enterococcus faecalis strain.

Tetracycline resistance heterogeneity in Enterococcus faecium.

The tetracycline (Tet) determinants, which encode resistance either to tetracyclines without minocycline (Tcr) or to tetracyclines including minocycline (Tcr-Mnr), of 30 wild-type clinical isolates of Enterococcus faecium were identified and localized. The Tet determinants were transferred by conjugation into a plasmid-free Enterococcus faecalis recipient at frequencies of 10(-6) to 10(-9) transconjugants per donor, as follows: Tcr, 6 strains; Tcr-Mnr, 14 strains; both Tcr and Tcr-Mnr, 6 strains; no detectable transfer, 4 strains. Classes L (Tcr phenotype) and M and O (Tcr-Mnr phenotype) of the Tet determinants were identified by DNA-DNA hybridization experiments.

Tn3702, a conjugative transposon in Enterococcus faecalis.

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.

Identification of chromosomal antibiotic resistance genes in Streptococcus anginosus ("S. milleri").

Determinants encoding resistance to antibiotics, except penicillin, of 5 of 21 Streptococcus anginosus clinical isolates that we examined transferred by conjugation into Enterococcus faecalis and into 1 to 5 streptococcal recipients at frequencies of 10(-6) to 10(-8) transconjugants per donor. No R plasmids were detected in any wild-type strain. DNA homology was detected between chromosomal fragments and the following genes: aadE in 7 strains that were highly resistant to streptomycin, aph-3 in 8 strains that were highly resistant to kanamycin, ermB in 11 of 15 strains that were resistant to erythromycin, and tetM in 14 and tetO in 3 of the 18 strains that were resistant to tetracycline-minocycline.

Does a tetracycline resistance determinant of class N exist?

pMV120 was reported to carry the tetracycline resistance (Tcr) determinant of class N. We obtained tetracycline-susceptible transconjugants harboring plasmids with restriction enzyme profiles indistinguishable from those of pMV120 isolated from tetracycline-resistant clones. We conclude that pMV120 is a cryptic plasmid and that class N of Tcr determinants does not exist.

High-level chromosomal gentamicin resistance in Streptococcus agalactiae (group B).

This is the first report of high-level gentamicin resistance in a group B streptococcus. Strain B128 of serotype II was isolated from an infected leg wound in 1987. B128 was resistant to high levels of gentamicin as well as of all other available aminoglycosides and was also resistant to tetracyclines.