In vivo development of tigecycline resistance in Klebsiella pneumoniae owing to deletion of the ramR ribosomal binding site

- Knowledge on the mechanisms underlying in vivo development of tigecycline (TIG) resistance is limited.
- Two TIG-susceptible (KP-1S, KP-2S) and two TIG-resistant (KP-3R, KP-4R) K. pneumoniae were isolated from the same patient.
- KP-3R and KP-4R were derived from KP-1S or KP-2S as a result of TIG exposure.
- ramR RBS deletion in KP-3R/4R that abolished ramR translation was identified as the mechanism contributing to TIG resistance.

Tigecycline resistance is emerging among Klebsiella pneumoniae, but knowledge regarding in vivo development of tigecycline resistance is limited. Here we report a new mechanism of tigecycline resistance in K. pneumoniae that evolved during tigecycline therapy. Klebsiella pneumoniae isolates were consecutively obtained from urine samples of a patient with scrotal abscess and urinary tract infection before and during tigecycline treatment. Two tigecycline-resistant K. pneumoniae strains (KP-3R and KP-4R; MIC = 8 µg/mL) were isolated after 41 days and 47 days of tigecycline therapy. These isolates had the same sequence type (ST11) and PFGE patterns as tigecycline-susceptible strains (KP-1S and KP-2S; MIC = 2 µg/mL) initially isolated from the patient. Compared with KP-1S and KP-2S, KP-3R and KP-4R exhibited higher expression of efflux pump AcrAB. Sequence comparison of the repressor gene ramR did not find any mutation within the open-reading frame that exist frequently in tigecycline-resistant K. pneumoniae. Instead, a 12-bp deletion of ramR upstream region including the ribosomal binding site (RBS) TGAGG was observed in KP-3R and KP-4R. qRT-PCR and immunoblotting analyses showed that KP-3R and KP-4R did not have impaired ramR transcription but had abolished RamR protein production. Furthermore, xylE reporter assay demonstrated that KP-3R and KP-4R had a defect in RamR translation caused by the 12-bp deletion. Complementing KP-3R and KP-4R with functional ramR suppressed expression of acrAB and consequently restored tigecycline susceptibility. This is the first report identifying deletion of the ramR RBS as a mechanism of in vivo tigecycline resistance in K. pneumoniae developing during tigecycline therapy.