A Smooth-Type, Phage-ResistantKlebsiella pneumoniaeMutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3

ABSTRACTBacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism ofKlebsiella pneumoniaewas revealed. After phage GH-K3 treatment, a smooth-type colony, named K7RB, was obtained from theK. pneumoniaeK7 culture. Treatment with IO4−and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7RBwere quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7RB.trans-Complementation of OmpC in K7RBconferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation ofompCin K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the nativeK. pneumoniaestrains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpCK7was the most suitable receptor for GH-K3. This study revealed that K7RBbecame resistant to GH-K3 due to gene mutation ofompCand that OmpC of K7 is essential for effective infection by GH-K3.IMPORTANCEWith increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to ourtrans-complementation,ompCmutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor ofK. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such asEscherichia coliandSalmonella, but also forK. pneumoniae.