Klebsiella pneumoniae is an opportunistic pathogen posing an urgent threat to global public health, and the capsule is necessary for K. pneumoniae infection and virulence. Phage-derived capsule depolymerases have shown great potential as antivirulence agents in treating carbapenem-resistant K. pneumoniae (CRKP) infections. However, the therapeutic potential of phages encoding depolymerases against CRKP remains poorly understood. In this study, we identified a long-tailed phage SRD2021 specific for mucoid CRKP with capsular K47 serotype, which is the predominant infectious K-type in Asia. Genome sequencing revealed that ΦSRD2021 belonged to the Drulisvirus genus and exhibited a capsular depolymerase domain in its tail fiber protein. A transposon-insertion library of host bacteria was constructed to identify the receptor for ΦSRD2021. We found that most phage-resistant mutants converted to a nonmucoid phenotype, including the mutant in wza gene essential for capsular polysaccharides export. Further knockout and complementation experiments confirmed that the Δwza mutant avoided adsorption by ΦSRD2021, indicating that the K47 capsular polysaccharide is the necessary receptor for phage infection. ΦSRD2021 lysed the bacteria mature biofilms and showed a therapeutic effect on the prevention and treatment of CRKP infection in the Galleria mellonella model. Furthermore, ΦSRD2021 also reduced the colonized CRKP in mouse intestines significantly. By recognizing the host capsule as a receptor, our results showed that ΦSRD2021 may be used as a potential antibacterial agent for K47 serotype K. pneumoniae infections.

Bile salts possess innate antibacterial properties and can cause significant damage to bacteria. To survive in the mammalian gut, Klebsiella pneumoniae has developed mechanisms to tolerate bile salts; however, the specific mechanisms remain unclear. Transposon library screening revealed that the efflux pump AcrAB is involved in bile salt resistance. acrA and acrB mutants exhibited high sensitivity not only to bile salts but also to SDS and various antibiotics, with a switch-loop, comprising residues G615, F616, A617, and G618, proving to be crucial in this process. A colonization defect of acrA and acrB mutants was demonstrated to be located in the mouse small intestine, where the bile salt concentration is higher compared to the large intestine. Additionally, both acrA and acrB mutants displayed reduced virulence in the Galleria mellonella model. In conclusion, our results suggest that the Resistance-Nodulation-Cell Division efflux pump serves as a critical determinant in the pathogenesis of K. pneumoniae through various aspects.

Colistin is regarded as an antibiotic of last resort against multidrug-resistant Gram-negative bacteria, including Klebsiella pneumoniae and Escherichia coli. Colistin resistance is acquired by microorganisms via chromosome-mediated mutations or plasmid-mediated mobile colistin resistance (mcr) gene, in which the transfer of mcr is the predominant factor underlying the spread of colistin resistance. However, the factors that are responsible for the spread of the mcr gene are still unclear. In this study, we observed that mcr-1 inhibited the transfer of the pHNSHP45 backbone in liquid mating. Similar inhibitory effect of mcr-1.6 and chromosomal mutant ΔmgrB suggested that colistin resistance, acquired from either plasmid or chromosomal mutation, hindered the transfer of colistin resistance-related plasmid in vitro. Dual plasmid system further proved that co-existing plasmid transfer was reduced too. However, this inhibitory effect was reversed in vivo. Some factors in the gut, including bile salt and anaerobic conditions, could increase the transfer frequency of the mcr-1-containing plasmid. Our results demonstrated the potential risk for the spread of colistin resistance in the intestine, provide a scientific basis against the transmission of colistin resistance threat.

Bacterial chemotaxis is the phenomenon in which bacteria migrate toward a more favorable niche in response to chemical cues in the environment. The methyl-accepting chemotaxis proteins (MCPs) are the principal sensory receptors of the bacterial chemotaxis system. Aerotaxis is a special form of chemotaxis in which oxygen serves as the signaling molecule; the process is dependent on the aerotaxis receptors (Aer) containing the Per-Arnt-Sim (PAS) domain. Over 40 MCPs are annotated on the genome of Vibrio cholerae; however, little is known about their functions. We investigated six MCPs containing the PAS domain in V. cholerae El Tor C6706, namely aer2, aer3, aer4, aer5, aer6, and aer7. Deletion analyses of each aer homolog gene indicated that these Aer receptors are involved in aerotaxis, chemotaxis, biofilm formation, and intestinal colonization. Swarming motility assay indicated that the aer2 gene was responsible for sensing the oxygen gradient independent of the other five homologs. When bile salts and mucin were used as chemoattractants, each Aer receptor influenced the chemotaxis differently. Biofilm formation was enhanced by overexpression of the aer6 and aer7 genes. Moreover, deletion of the aer2 gene resulted in better bacterial colonization of the mutant in adult mice; however, virulence gene expression was unaffected. These data suggest distinct roles for different Aer homologs in V. cholerae physiology.