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The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to  rpsL  mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by  rpsL  mutations in all 127 clinical strains of  Klebsiella pneumoniae  isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of  K. pneumoniae , nucleotide substitution or insertion located within the  rpsL  was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD 50  (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼1,000-fold less lethal (∼2×10 5  CFU) and the K87R mutant was ∼50-fold less lethal (∼1×10 4  CFU) than the parental strain (∼2×10 2  CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost ( P <0.01) in an  in vitro  growth competition experiment. In summary, most of the  K. pneumoniae  strains with streptomycin resistance caused by  rpsL  mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using  rpsL  as a counter-selection marker.

A Suitable Streptomycin-Resistant Mutant for Constructing Unmarked In-Frame Gene Deletions Using rpsL as a Counter-Selection Marker