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ABSTRACT Amikacin and colistin are effective against carbapenem-resistant Klebsiella pneumoniae. In 2017, we successively isolated three carbapenem-resistant K. pneumoniae isolates (ST967) from a patient with chronic renal failure in Japan. The first (SMKP01, sputum, day 0) and second (SMKP02, blood, day 14) strains were resistant to most antimicrobials tested but still susceptible to amikacin (MICs of 4 and 0.5 mg/liter, respectively) and colistin (MIC of 0.5 mg/liter for both). The third strain (SMKP03, blood, day 51) was not susceptible to amikacin (MIC, 32 mg/liter), and its MIC for colistin varied (0.5 to 8 mg/liter). Whole-genome sequencing of SMKP01 revealed that 17 of 20 antimicrobial resistance genes, including qnrB91 (a novel qnrB2 variant) and aac(6′)-Ib-cr, were located on an 86.9-kb IncFII-IncQ plasmid. The qnrB91 conferred greater fluoroquinolone resistance than qnrB2. SMKP03 aac(6′)-Ib-cr that possessed a gene mutation that resulted in an R102W substitution, namely, aac(6′)-Ib-D179Y, made a greater contribution to amikacin resistance than did aac(6′)-Ib-cr. SMKP03 harbored a nonsense mutation in mutS, which encodes a DNA repair enzyme. Introduction of this mutation into SMKP01 (SMKP01mutSA307T) resulted in a dramatic increase (>58-fold) in the frequency of spontaneous amikacin-resistant mutants relative to SMKP01, and the substantial mutants possessed aac(6′)-Ib-D179Y. SMKP01mutSA307T exhibited an unstable MIC for colistin (0.5 to 8 mg/liter). The results demonstrate that a disruptive mutation in MutS, arising during the clinical course of an infection, created a platform for the acquisition of amikacin nonsusceptibility and colistin heteroresistance in multidrug-resistant K. pneumoniae, mediated by the elevated frequency of spontaneous mutations. IMPORTANCE The emergence of multidrug resistance in pathogens such as Klebsiella pneumoniae is of great clinical concern. Antimicrobial resistance sometimes arises during the course of an infection. Although many studies have reported the emergence of antimicrobial resistance and novel antimicrobial resistance genes in the clinical isolates, the identity of the bacterial factor(s) that generate this emergence is still unclear. We report that a disruptive mutation in MutS, arising during the clinical course of an infection, created a context for the acquisition of colistin resistance and the emergence of a novel variant of the amikacin resistance gene in multidrug-resistant K. pneumoniae via an increase in the frequency of spontaneous mutation. This observation is important for understanding how K. pneumoniae develops multidrug resistance during infection and could potentially lead to new antimicrobial treatments for high-risk pathological microbes.