We have analyzed the total metagenomic DNA from both human oral and fecal samples derived from healthy volunteers from six European countries to determine the molecular basis for tetracycline and erythromycin resistance. We have determined that tet(M) and tet(W) are the most prevalent tetracycline resistance genes assayed for in the oral and fecal metagenomes, respectively. Additionally, tet(Q), tet(O), and tet(O/32/O) have been shown to be common. We have also shown that erm(B), erm(V), and erm(E) are common erythromycin resistance genes present in these environments. Further, we have demonstrated the ubiquitous presence of the Tn916 integrase in the oral metagenomes and the Tn4451 and Tn1549 integrase genes within the fecal metagenomes.

ObjectivesTo identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient.MethodsAgar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay.ResultsThe tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46).ConclusionsThis is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota.

Enterococcus faecium 664.1H1 is multiply antibiotic resistant and mercury resistant. In this study, the genetic support for the tetracycline resistance of E. faecium 664.1H1 was characterized. The tet(S) gene is responsible for tetracycline resistance, and this gene is located on the chromosome of E. faecium 664.1H1, on a novel conjugative transposon. The element is transferable to Enterococcus faecalis, where it integrates into a specific site. The element was designated EfcTn1. The integrase of EfcTn1 is related to the integrase proteins found on staphylococcal pathogenicity islands. We show that the transposon is flanked by an 18-bp direct repeat, a copy of which is also present at the target site and at the joint of a circular form, and we propose a mechanism of insertion and excision.