Xiao-Jun Li

Background: Klebsiella pneumoniae frequently causes hospital-acquired and community-acquired infections, posing a serious threat to human health. Lytic phages can rapidly propagate within host bacterial cells and release progeny phages, ultimately inducing the death of the host bacteria. K. pneumoniae depolymerases are enzymes capable of specifically degrading bacterial surface polysaccharides, thereby disrupting bacterial biofilms, which offers a new approach to the prevention and control of K. pneumoniae . Methods: Morphology of phage vB_KpnP_JYSS5 was observed through transmission electron microscopy. The optimal multiplicity of infection, adsorption curve, one-step growth curve, and temperature and pH stability were determined using the double-layer agar plate method. Whole-genome sequencing was performed on the DNBSEQ-T7 sequencing platform. The putative depolymerase gene (ORF11) from the phage genome was subjected to prokaryotic expression. The anti-biofilm activity of the phage and the depolymerase was assessed using plate counting, scanning electron microscopy, and confocal laser scanning microscopy. The effects of depolymerase treatment on macrophage phagocytic function were evaluated using plate counting, ELISA, and flow cytometry. A mouse infection model was established by intraperitoneal injection of K. pneumoniae to assess the efficacy of the depolymerase. Results: Phage vB_KpnP_JYSS5 specifically lyses the K57-type K. pneumoniae strain 21AA3004. It is a lytic phage with a DNA genome of 40,795 bp. The optimum MOI is 0.001. It has an average burst size of approximately 114 PFU/cell. The phage retained its infectivity between 4 °C and 60 °C and at a pH range of 5 to 11. The phage effectively inhibited biofilm formation and degraded pre-existing biofilms of host strain. Encoded by ORF11, Dep11 effectively degraded the capsule, suppressed biofilm formation, and eliminated mature biofilms but also potentiated polymyxin B and gentamicin against bacteria. Dep11 enhanced macrophage phagocytosis. In animal experiments, Dep11 (50 µg) raised the survival rate of infected mice to 70% and lowered four pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ) in their lung tissue. Conclusion: Phage vB_KpnP_JYSS5 is a novel double-stranded DNA phage classified within the order Autographivirales . Depolymerase Dep11 specifically targets the capsular polysaccharide of K57 K. pneumoniae , making it a promising therapeutic agent for the treatment of K57 K. pneumoniae infections.

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a major nosocomial pathogen, poses an increasingly serious threat to human health. In the quest for innovative therapeutic modalities to treat CRKP infections, a newly discovered phage, vB_KpnM_JYSS3, which can specifically infect and lyse K2-type CRKP, was isolated and subjected to an array of comprehensive characterizations and genome sequencing. Furthermore, an analysis of the expression of the tail protein (ORF17) and the detection of its functions were carried out. Double-layer agar plates containing CRKP were used to isolate phages from hospital sewage samples. The phage's optimal multiplicity of infection (MOI), lytic spectrum, bactericidal activity, stability, and one-step growth curve were tested. The biofilm development ability of the CRKP strain 21AA2216 and the phage antibiofilm activity were determined using a 96-well microtitration plate. vB_KpnM_JYSS3 prevented biofilm generation and broke down mature biofilms. The whole-genome sequencing results revealed that vB_KpnM_JYSS3 is a toxic phage (nontemperate). The genome size of vB_KpnM_JYSS3 is 47,244 bp, and the G + C content is 48.48 mol%. A total of 71 coding sequences (CDSs) were predicted. Protein function prediction revealed that the tail protein (ORF17) of vB_KpnM_JYSS3 may possess depolymerase activity, consistent with the halo phenomenon around the plaque. ORF17 was expressed, purified, and named Dep17. Dep17 can degrade the capsular polysaccharide (CPS) surrounding K2-type CRKP, and it can also enhance the activity of polymyxin B against K2-type CRKP biofilms. Phage vB_KpnM_JYSS3 and its novel depolymerase Dep17 may represent promising strategies for combating infections resulting from biofilm-forming and drug-resistant K2-type CRKP.

Background: Integrated traditional Chinese medicine and biomedicine is an effective method to treat tuberculosis (TB). In our previous research, traditional Chinese medicine preparation NiuBeiXiaoHe (NBXH) achieved obvious anti-TB effects in animal experiments and clinical practice. However, the action mechanism of NBXH has not been elucidated.Method: Peripheral blood mononuclear cells (PBMCs) were collected to extract mRNA and differentially expressed (DE) genes were obtained using gene microarray technology. Finally, GEO databases and RT-qPCR were used to verify the results of expression profile.Result: After MTB infection, most upregulated DE genes in mice were immune-related genes, including cxcl9, camp, cfb, c4b, serpina3g, and ngp. Downregulated DE genes included lrrc74b, sult1d1, cxxc4, and grip2. After treatment with NBXH, especially high-dose NBXH, the abnormal gene expression was significantly corrected. Some DE genes have been confirmed in multiple GEO datasets or in pulmonary TB patients through RT-qPCR.Conclusion: MTB infection led to extensive changes in host gene expression and mainly caused the host’s anti-TB immune responses. The treatment using high-dose NBXH partially repaired the abnormal gene expression, further enhanced the anti-TB immunity included autophagy and NK cell-mediated cytotoxicity, and had a certain inhibitory effect on overactivated immune responses.