Molecular ecology of tetracycline resistance: Development and validation of primers for detection of tetracycline resistance genes encoding ribosomalprotection proteins

Phylogenetic analysis of tetracycline resistance genes encoding the ribosom al protection proteins (RPPs) revealed the monophyletic origin of these gen es. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W,Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on th is phylogenetic analysis, a set of PCR primers for detection, retrieval, an d sequence analysis of the corresponding gene fragments from a variety of b acterial and environmental sources was developed and characterized. a pair of degenerate primers targeted all tetracycline resistance genes encoding R PPs except otrA and tet, and seven other primer pairs were designed to targ et the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine f ecal streptococci. Classes Tet O and Tet W were found in the intestinal con tents of both animals, while Tet hi was confined to pigs and Tet Q was conf ined to the rumen. The tet(O) and tet(W) genes circulating in the microbiot a of the rumen and the gastrointestinal tract of pigs were identical despit e the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carr ied tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classe s of RPPs except Tet T and TetB P were found in the commercial components o f swine feed. This is the first demonstration of the applicability of molec ular ecology techniques to estimation of the gene pool and the flux of anti biotic resistance genes in production animals.