Abstract
ABSTRACTTetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one.This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm.
Highlights
Since their discovery in the 1940s, tetracyclines have been used for many years to treat a wide spectrum of infections
Some tetracyclines are still useful in some treatments, such as in acne vulgaris, periodontitis and infections caused by multidrug resistant (MDR) Acinetobacter baumannii, Helicobacter pylori and methicillin-resistant Staphylococcus aureus [2,3,4]
According to http://faculty.washington.edu/marilynr/, 60 tetracycline resistance genes have been detected that code for these mechanisms, along with 11 mosaic genes that code for ribosomal protection proteins
Summary
Subgingival samples were collected from periodontally healthy volunteers in the dental clinic of the Universitat Internacional de Catalunya (UIC), (Barcelona, Spain), and the research protocol was approved by the Ethics Committee of the UIC (study number: PER-ECL -2011–06-NF). DNA was extracted from all the isolates using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, quantified using a Nanodrop 2000 UV-vis spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and visualized in a 0.5% agarose gel stained with ethidium bromide. The PCR products were purified using the E.Z.N.A.® Gel extraction Kit (Omega BIO-TEK, Norcross, GA, USA) and sequenced by primer walking at the Genomics and Bioinformatics Service of the Autonomous University of Barcelona (Barcelona, Spain). Primers tetB-insert-F and tetB-insert-R (Table 1) were used to fully sequence the tet(B) gene. RNA was extracted from liquid cultures with and without tetracycline using the High Pure RNA Isolation kit (Roche Diagnostics, Mannheim, Germany) according to the instructions provided by the manufacturer. The absence of DNA in the samples was verified by PCR using the sets of primers tetB-RT-F/tetB-RT-R, and 16S-So-F/16S-So-R (Table 1), visualizing the results in a 3% agarose gel. Streptococcus pneumoniae ATCC 49619 was used as a quality control strain
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