The class L (TetL) tetracycline resistance determinant from streptococci specified resistance and an energy-dependent decreased accumulation of tetracycline in both Streptococcus faecalis and Escherichia coli. Using E. coli, we showed that the reduced uptake resulted from active efflux. The streptococcal class M determinant, known to render the protein synthesis machinery of S. faecalis resistant to tetracycline inhibition, did not alter tetracycline transport in either host.Tetracycline resistance (Tcr) determinants are widespread among gram-negative and gram-positive bacteria, both anaerobes and aerobes (10). The original source of these determinants is unknown, but recent studies have shown that previously identified determinants appear to have moved into new hosts. Of particular note, determinants with strong homology to class M (TetM), initially found among streptococci (3), have now been identified among newly appearing Tcr strains of Mycoplasma (17), Ureaplasma (16), Campylobacter (19), Gardnerella (15), Neisseria (14), and Clostridium (7) spp. The wide dispersal of this determinant can probably be attributed in part to its existence on transposon Tn916 originally described in Streptococcus faecalis (5). Two other streptococcal determinants, class L (TetL) and class N (TetN), have also been discovered (3).The mechanism of resistance specified by the class M determinant in S. faecalis occurs at the level of the target of tetracycline, namely, the protein synthesis machinery within the cell (2). The class L determinant, on the other hand, does not prevent the inhibition of protein synthesis in S. faecalis but rather decreases tetracycline uptake (2). The purpose of the present study was to determine the mechanism for this decrease in both S. faecalis and Escherichia coli as hosts.The naturally occurring plasmids pMV158 (class L) and pAM211 (class M) (2) were used in S. faecalis. In E. coli, we used pVB A15 (a hybrid of pMV158 with the E. coli cloning vector pVH2124 [3]) and pJI3 (a 5-kilobase HinclI region of the chromosomal class M determinant from Streptococcus agalactiae B109 containing the entire Tcr determinant cloned into the E. coli vector pACYC177 [3,8]). These plasmids were introduced into E. coli SK1592 (3) by transformation.The levels of resistance to tetracycline and minocycline expressed aerobically and anaerobically in S. faecalis and E. coli were assayed at 37°C by the gradient plate method on Penassay medium (Difco Laboratories) (4). In both genera, the class M determinant specified resistance to both tetracycline and minocycline, while the class L determinant, as previously reported for streptococci (2), expressed only resistance to tetracycline. In S. faecalis, Tcr mediated by class L on pMV158 varied (MIC, 50 to 100 ,ug/ml) in different experiments and that specified by class M on pAM211 was
Escherichia coli bearing a cryptic tetracycline resistance determinant from Bacteroides fragilis expressed low-level constitutive resistance to tetracycline under aerobic, but not anaerobic, growth conditions and accumulated less tetracycline aerobically than did isogenic susceptible cells. This decreased uptake was energy dependent and reversible by increased concentrations of tetracycline, suggesting a saturable carrier-mediated active efflux mechanism. Decreased uptake was not seen when the cells were grown and assayed anaerobically. Other tetracycline resistance determinants (classes A to E) isolated from gram-negative enteric bacteria expressed resistance and generated active efflux of tetracycline under anaerobic as well as aerobic conditions. When the Bacteroides determinant was placed in the same cell with any of the class A to E tetracycline resistance determinants, there was an increase in resistance under aerobic conditions of as much as 48% more than was projected by adding the resistances expressed by the determinants individually. In cells bearing the class A determinant together with the Bacteroides determinant, saturation of the active efflux system required over twofold more exogenous tetracycline than did ceUs bearing the class A determinant alone. We have designated this new tetracycline resistance determinant class F.Tetracycline resistance (Tc9 in members of the family Enterobacteriaceae and other facultative aerobic gramnegative bacteria is encoded by at least five classes (A to E) of resistance determinants showing different levels of resistance to tetracycline and its analogs (8,10,12,16). Although the classes can be distinguished under stringent DNA hybridization conditions (16), low-stringency hybridization and DNA sequence analysis of the determinants so far studied indicate that they are very similar and seem to have evolved from a common origin (7,12,22). Recently, a cryptic Tcr determinant was discovered when a fragment containing the clindamycin resistance determinant from Bacteroides fragilis was cloned in Escherichia coli (5). Resistance was expressed during aerobic growth in the new host but not under anaerobic growth conditions. In this paper we demonstrate that the aerobic resistance specified by this determinant involves an energy-dependent saturable reduced uptake of tetracycline suggestive of an active efflux mechanism such as has been demonstrated for Tcr determinants among the Enterobacteriaceae (10, 15). Cells containing the Bacteroides determinant and any one of five other Tcr determinants on plasmids from the Enterobacteriaceae had a level of resistance higher than was projected by adding their respective resistances. The findings suggest that the products of the two determinants are interactive. The Bacteroides determinant defines a new class, TetF. (15) with and without plasmids were used for these studies. Plasmids bearing the different classes of Tcr determinants were RK231 and pIP15 (class A), R100-1 and R222 (class B), R144 and pBR322 (class C), RAl (class D), an...
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