Abstract
Klebsiella pneumoniae is a gram-negative bacterium that causes numerous diseases, including pneumonia and urinary tract infections. An increase in multidrug resistance has complicated the treatment of these bacterial infections, and although tigecycline shows activity against a broad spectrum of bacteria, resistant strains have emerged. In this study, the whole genomes of two clinical and six laboratory-evolved strains were sequenced to identify putative mutations related to tigecycline resistance. Of seven tigecycline-resistant strains, seven (100%) had ramR mutations, five (71.4%) had lon mutations, one (14.2%) had a ramA mutation, and one (14.2%) had an rpsJ mutation. A higher fitness cost was observed in the laboratory-evolved strains but not in the clinical strains. A transcriptome analysis demonstrated high expression of the ramR operon and acrA in all tigecycline-resistant strains. Genes involved in nitrogen metabolism were induced in the laboratory-evolved strains compared with the wild-type and clinical strains, and this difference in nitrogen metabolism reflected the variation between the laboratory-evolved and the clinical strains. Complementation experiments showed that both the wild-type ramR and the lon genes could partially restore the tigecycline sensitivity of K. pneumoniae. We believe that this manuscript describes the first construct of a lon mutant in K. pneumoniae, which allowed confirmation of its association with tigecycline resistance. Our findings illustrate the importance of the ramR operon and the lon and rpsJ genes in K. pneumoniae resistance to tigecycline.
Highlights
Klebsiella pneumoniae is a gram-negative bacterium of the Enterobacteriaceae family [1] that can cause numerous diseases, including pneumonia, urinary tract infections, septicemia, and pyogenic live abscesses [2]
Clinical strains and in vitro selection of mutants with tigecycline resistance We obtained two K. pneumoniae strains that were isolated from the blood of a patient during tigecycline treatment
We found that the MICs for tigecycline increased in a step-wise manner with the presence of mutations in the ramR operon and the lon and rpsJ genes
Summary
Klebsiella pneumoniae is a gram-negative bacterium of the Enterobacteriaceae family [1] that can cause numerous diseases, including pneumonia, urinary tract infections, septicemia, and pyogenic live abscesses [2]. Strains resistant to tigecycline have been reported [5,6,7,8]. RamA transcription is de-repressed by the ramR mutation in K. pneumoniae [5], and both rarA and marA provide alternate pathways for RamA-independent tigecycline resistance [11]. We combined whole-genome sequencing and RNA-Seq to identify putative mutations related to tigecycline resistance in both clinical and laboratory-evolved strains of K. pneumoniae. Mutations in the ramR, lon, ramA and rpsJ genes were observed in the tigecycline-resistant strains. A transcriptome analysis demonstrated that the ramR locus was highly expressed in all tigecycline-resistant strains. To confirm the role of ramR and lon in tigecycline resistance in K. pneumoniae, we performed a complementation experiment and constructed a knockout strain
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