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Using colony hybridization techniques and DNA probes derived from four distinct tetracycline resistance determinants, we have examined the frequency of these determinants among 225 lactose-fermenting coliforms isolated from fecal samples of both humans and animals. The class B, or TnJO-type determinant, occurred most frequently at 73.3%, followed by class A (on RP1) at 21.7%, and class C (on pSC101) at 8%; 3.5% of isolates harbored two of these determinants. Hybridization to class D, carried by plasmid RA1, was not found among any of the isolates. One isolate failed to hybridize to any of the probes and represents a fifth class of determinant. No dramatic differences were observed in the frequencies of these determinants among four populations examined: hospital, urban, rural, and laboratory. At low stringency conditions of hybridization we were able to demonstrate cross-hybridization of determinant A with class C DNA and limited reaction with class B DNA, but no reaction with class D DNA.Tetracycline resistance, commonly specified by plasmids, is widely disseminated among various bacterial species and is expressed at different levels of resistance (11, 12). We and others have used the tetracycline analogs chelocardin and minocycline to demonstrate phenotypic differences among strains carrying tetracycline resistance (12, 13). Such evidence strongly suggested genetic heterogeneity of these determinants, and this was later confirmed by DNA-DNA hybridization with unique probes for three different phenotypic resistance determinants (12). Subsequent evidence has demonstrated an even greater heterogeneity among tetracycline resistance determinants; there appears to be no detectable homology between those found in gram-negative and gram-positive species (3,14). Recently, we have developed a specific probe for the tetracycline determinant classified as class D. Using this and probes for the other three determinants, we examined the frequency of classes A through D among 225 tetracyclineresistant, lactose-fermenting coliforms isolated from 199 fecal samples of human and animal donors. All but one of the isolates hybridized to one or more of the known tetracycline resistance determinants. The results support the uniqueness of the four determinants and demonstrate a marked difference in the frequency of these determinants among fecal coliforms. This study t Present address:

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Oxytetracycfine-resistant (OTcr) and tetracycline-resistant (Tc') Aeromonas hydrophila were isolated commonly from catfish intestinal contents and the water and sediment of catfish culture ponds, but less frequently in market catfish. Isolates demonstrated two resistance phenotypes, Tcr OTcr and Tcs OTcr, when plated directly on to MacConkey agar containing 30 Fig of tetracycline or oxytetracycline per ml. Tcs OTcr isolates expressed Tcr after induction by 1 h of growth in tryptic soy broth containing 1 p.g of tetracycline per ml, Over 90% of the resistant aeromonads hybridized with DNA probes for class A or class E Tcr determinants; class E was twice as prevalent as class A. The distribution of class A and E Tcr determinants varied with the Tcr phenotypes. Prior to induction, 86% of isolates with class A determinants were Tcr as well as OTcr, while only

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

The tetracycline resistance determinant on plasmid pVM111 from an avian strain of Pasteurella multocida mediates tetracycline resistance by a regulated active efflux mechanism. DNA coding for the determinant did not hybridize at high stringency with DNA representing a group of common tetracycline resistance determinants. The DNA sequence, however; revealed a structural gene and a repressor gene which had significant (37 to 64%) sequence similarities with previously described classes of efflux-type tetracycline resistance genes from members of the family Enterobacteriaceae. The new determinant has been assigned to class H.

The structural and regulatory tetracycline resistance genes of transposon Tn10 are located on a 2,700-base pair HpaI fragment. We have used eight tetracycline-sensitive mutations in the 2,700-base pair fragment, cloned into two compatible plasmids, to demonstrate that two complementation groups are required for tetracycline resistance. By genetic recombination with plasmids containing the regulatory or structural regions for resistance, we have determined that both complementation groups reside within the structural region. The complementation groups, designated tetA and tetB, are proximal and distal, respectively, to the promoter for the tetracycline resistance structural region. The tetB mutations are in the portion of the structural region that is known to encode the 36,000-molecular-weight, inner-membrane TET protein. The levels of tetracycline resistance expressed during complementation suggest a complex interaction between the products of the tetA and tetB loci.