Molecular Ecology of Tetracycline Resistance: Development and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins

Abstract

Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. 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 this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target 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 fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals.

Macrolide and Tetracycline Resistance andemmType Distribution ofStreptococcus pyogenesIsolates Recovered from Turkish Patients

“…3,10 In some studies, tetracycline resistance in S. pyogenes was found to be associated with the tetM gene and, less frequently, with the tetO gene. 16,29,34 Since tetracycline resistance genes can reside in mobile genetic elements that carry macrolide resistance genes, the cooccurrence of resistance to both classes of drugs is possible.…”

“…36 Primer sets specific for the several genes were used as seen in Table 1. 3,16 Two reference Escherichia coli strains carrying erm(A) and erm(B) genes and one S. pyogenes strain positive for mef(A) (kindly provided by Dr. Ziya Cibali Acikgoz) were used as positive PCR controls.…”

Abundance and Diversity of Tetracycline Resistance Genes in Soils Adjacent to Representative Swine Feedlots in China

“…All PCR assays were conducted in a 25 µL volume reaction, using a Peltier Thermal Cycler (Bio-Rad, Hercules, CA (30,33), and their sequences are listed in Table S1 (Supporting Information). The temperature program was initially denaturated at 94°C for 4 min, followed by 35 cycles of 45 s at 94°C, 45 s at different annealing temperatures (Table S1), and 72°C for 1 min, with a final extension step for 6 min at 72°C.…”

“…Among the tet genes, tetM is known to have the widest host range of any tet genes (16) since it is often associated with mobile genetic elements which enhance its transferability from one bacteria to another (27). Several studies have reported a variety of genotypes of the tetM from natural environments (28,29) or resistance isolates (10,30), and various genotypes of the tetM are known to occur in human and animal pathogens (29).…”

Safety Evaluation Of Sjenica Cheese With Regard To Coagulase-Positive Staphylococci And Antibiotic Resistance Of Lactic Acid Bacteria And Staphylococci

“…As the fi rst step, we used tetracycline resistance genes encoding the ribosomal protection proteins (RPPs). As phylogenetic analysis confi rmed the monophyletic origin of these determinants, it is possible to design a set of PCR primers which in positive samples give the amplicons of RPP genes in general [70]. After the isolates were subjected to PCR amplifi cation with the universal primer set, we exposed the tested isolates to PCR screening with classspecifi c primers Tet M and Tet K. Tet K protein revealed the different mechanisms of tetracycline resistance-i.e.…”