Copresence of tet(K) and tet(M) in Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Is Associated with Increased Fitness during Exposure to Sublethal Concentrations of Tetracycline

Abstract

The tetracycline resistance gene tet(K) was shown to be integrated within the predominant staphylococcal cassette chromosome mec (SCCmec) element of Danish livestock-associated methicillin-resistant Staphylococcus aureus CC398 (LA-MRSA CC398). These LA-MRSA CC398 isolates already possessed tet(M), but the acquisition of tet(K) significantly improved their fitness at sublethal concentrations of tetracycline. Because tet(K) is genetically linked to SCCmec, the use of tetracycline in food animals may have contributed to the successful spread of LA-MRSA CC398.

FIG 1

Organization of tet(K) promoters. (A) Integrated and free forms of pT181 in SCCmec Vc-bearing isolates. (B) Free form of pT181 in isolates harboring nonsubtypeable SCCmec V elements. The solid boxes represent −10 and −35 sequences of the promoters P_hybrid, P_out, and P_tet(K). The arrows above the sequences represent transcription start points. The arrows underneath the sequences represent the start of IS257 and the end of repC, respectively. The imperfect 27-bp terminal inverted repeat bounding IS257 is underlined. Lowercase letters represent the 8-bp target duplication. See reference for further details.

FIG 2

Comparison of the growth rates of six tet(K)-positive and six tet(K)-negative LA-MRSA CC398 isolates. The isolates are described in Table S1 in the supplemental material. Single cultures of each isolate were grown overnight in MHB, diluted to an optical density at 600 nm of 0.07 in 200 μl fresh MHB containing 0, 0.5, 1, 2, 4, 8, 16, or 32 mg/liter tetracycline, and grown at 37°C in a PowerWave 340 reader (BioTek, Winooski, VT). The OD₆₀₀ was measured over time and used to calculate the number of divisions per minute during exponential growth. The statistical differences between the growth rates were determined using the unpaired two-tailed Student's t test (GraphPad Prism software version 5.0; GraphPad Software, Inc., San Diego, CA). Abbreviations: ns, not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.