New tetracycline resistance determinants coding for ribosomal protection in streptococci and nucleotide sequence of tet(T) isolated from Streptococcus pyogenes A498

Abstract

An approach based on PCR has been developed to identify new members of the tet gene family in streptococci resistant to tetracycline and minocycline. Degenerate primers, corresponding to portions of the conserved domains of the proteins Tet(M), Tet(O), TeTB(P), Tet(Q), and Tet(S), all specifying the tetracycline-minocycline resistance phenotype, were used to selectively amplify DNA fragments within the coding sequences. Nine streptococcal strains which do not carry the genes tet(M), tet(O), tetB(P), tet(Q), or tet(S) were investigated. Four of them gave no detectable PCR products. The five remaining strains each yielded a PCR product of 1.1 kbp. DNA hybridization experiments showed that these putative Tet determinants fell into four new hybridization classes, of which one, Tet T, was further analyzed. The gene tet(T) was isolated from Streptococcus pyogenes A498, and the nucleotide sequence that was necessary and sufficient for the expression of tetracycline resistance in Escherichia coli was determined. The deduced Tet(T) protein consists of 651 amino acids. The protein most closely related to Tet(T) was Tet(Q), which has 49% identical amino acid residues. A phylogenetic analysis revealed that Tet T represents a novel branching order among the Tet determinants so far described.

Antibiotic survey ofLactococcus lactisstrains to six antibiotics by Etest, and establishment of new susceptibility-resistance cut-off values

“…DNA dilutions were used as template in PRC reactions with universal primers of ribosomal protection genes (DI, (Clermont et al 1997). Following PCR conditions as described by Gevers et al (2003), specific primers for tet(K) (TetK-FW1 5k-TTATGGTGGTTGTAGCTAGAAA-3k and TetK-RV1 5k-AAAGGGTTAGAAACTCTTGAAA-3k), tet(L) (TetL-FW3 5k-GYMHYYHVHVHVYSYSYYVV-3k and TetL-RV3 5k-GTGAAMGRWAGCCCACCTAA-3k), and tet(M) (DI and tetMR, 5k-CACCGAGCAGGGATTTCTC-CAC-3k) were also used.…”

Macrolide, lincosamide and tetracycline susceptibility and emm characterisation of invasive Streptococcus pyogenes isolates in Israel

“…In Turkey, ERY resistance was observed in 2.7% of isolates (this low rate being ascribed to the limited use of ERY, especially for streptococcal pharyngitis), whilst 22.7% were resistant to TET [42]. Although TET resistance in other bacteria has been studied extensively [43], less is known about the resistance mechanism in GAS [5,[17][18][19][20][21][22][23]. According to current knowledge, the mechanisms of resistance are mainly associated with the presence of the tet(M) or tet(O) genes, which are related to transposon elements.…”

“…Indeed, erythromycin (ERY) resistance in GAS has been the subject of many reports in recent years [4,[6][7][8][9][10][11][12][13][14][15][16]. In contrast, less information is available on the characteristics of TET resistance mechanisms [5,[17][18][19][20][21][22][23] despite the fact that TET resistance has been known for decades [24] and has been recorded in upwards of 50% of isolates [21]. Variation in the prevalence of resistance to these antibiotics has been attributed to clonality of resistant isolates and to particular practices of antibiotic use in different countries [21,[25][26][27].…”

Phenotypic, genetic and technological characterization of Lactococcus garvieae strains isolated from a raw milk cheese

“…The presence of tetracycline resistance genes was checked by PCR using the universal primers for genes encoding ribosomal protection proteins DI (5 0 -GAYACICCIGGICAYRTIGAYTT-3 0 ) and DII (5 0 -G CCCARWAIGGRTTIGGIGGIACYTC-3 0 ) (Clermont, Chesneau, de Cespedes, & Horaud, 1997), and following the reported PCR conditions. Amplicons were purified and sequenced by cycle extension in an ABI 370 DNA sequencer (Applied Biosystems, Foster City, CA, USA).…”

“…Amplification of tetracycline resistance genes with the universal primers DI and DII(Clermont et al, 1997) for ribosomal protection genes from L. garvieae strains. Order: Lanes 1-11, order of the strains as in the tables (T1-1, T1-41, T2-4, T2-17, T3-18, T3-22, Q1-21, Q2-41, CECT 4531 T , LMG 6890 T , and NCIMB 700608); Cþ, positive amplification control (DNA from Lactococcus lactis AA29, which carries a functional tet(M) gene; Fló rez, Ammor, & Mayo, 2008); B, blank to which DNA was not added; M, Gene Ruler ExpressÔ DNA ladder (Fermentas Gmbh., Germany); the molecular weight (kbp) of some bands is indicated on the left.…”