High Prevalence of Klebsiella pneumoniae Infections in AnHui Province: Clinical Characteristic and Antimicrobial Resistance.

BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) causes community-acquired and hospital-acquired pneumonia. The mortality rates of invasive infections caused by hypervirulent K. pneumoniae (HvKP) are extremely high. However, the microbiological characteristics and clinical manifestations of K. pneumoniae in AnHui province still remain unclear. PURPOSE: To show the high prevalence of HvKP infections regarding clinical characteristics and antimicrobial resistance in Anhui province. PATIENTS AND METHODS: A retrospective analysis was conducted to study the clinical data of 115 strains of K. pneumoniae from July 2019 to March 2020 in The First Affiliated Hospital of AnHui Medical University. The virulence genes, capsular types, carbapenemase genes, and molecular subtypes of these hypervirulent isolates were detected. RESULTS: Overall, 59.1% (68/115) cases were HvKP infections, mainly from the department of intensive care unit (ICU, n=14, 20.6%) and the department of respiratory and critical care (n=13, 19.1%). K2 was the most prevalent capsular serotype (n=26), followed by K1 (n=21). The results of MLST identification of 68 strains showed that ST23 (n=15, 22.1%) was the most common type of ST, followed by ST11 and ST65 (n=12, 17.6%), ST86 (n=9, 13.2%), and ST412 (n=6, 8.8%). Among 68 hvKP strains, 12 isolates were carbapenem resistant, and all except two harboured KPC. CONCLUSION: The high incidence of carbapenemase producing HvKP in the Anhui province, especially the higher mortality of HvKP, should be paid more attention. Meanwhile, epidemiological surveillance and clinical treatment strategies should be continuously determined and implemented.

Microbiota-Derived Short-Chain Fatty Acids Promote LAMTOR2-Mediated Immune Responses in Macrophages.

ABSRTACTKlebsiella pneumoniae is a common cause of human-pneumonia-derived sepsis with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis. The mechanisms by which the gut microbiota affects the host defenses in the respiratory system systematically, however, remain poorly understood. Here, we show that gut microbiota depletion increases susceptibility to extracellular K. pneumoniae infections in terms of increased bacterial burdens in lung and decreased survival rates. Oral supplementation with gut microbiota-derived short-chain fatty acids (SCFAs), subsequently activating G protein-coupled receptor 43 (GPCR43), enhances a macrophage's capacity to phagocytose invading K. pneumoniae Furthermore, SCFAs and GPR43 increase macrophage bacterial clearance by upregulating LAMTOR2, which is further identified as an antibacterial effector and elucidated to facilitate phagosome-lysosome fusion and extracellular signal-regulated kinase (ERK) phosphorylation. Lastly, conditional ablation of Lamtor2 in macrophages decreases their antimicrobial activity, even though mice were pretreated with exogenous SCFA supplementation.IMPORTANCE These observations highlight that SCFAs promote macrophage elimination of K. pneumoniae via a LAMTOR2-dependent signal pathway and suggest that it is possible to intervene in K. pneumoniae pneumonia by targeting the gut microbiota.

Alterations in the Gut Microbiome and Cecal Metabolome During Klebsiella pneumoniae-Induced Pneumosepsis.

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis in human and is associated with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis during bacterial infections. However, the mechanisms by which the gut microbiota affects immune responses in the lung still remain poorly understood. Here, we performed cecal metabolomics sequencing and fecal 16s rRNA sequencing in K. pneumoniae-infected mice and uninfected controls and showed that K. pneumoniae infection led to profound alterations in the gut microbiome and thus the cecal metabolome. We observed that the levels of Lactobacillus reuteri and Bifidobacterium pseudolongum were significantly decreased in K. pneumoniae-infected mice. Spearman correlation analysis showed that alterations in the richness and composition of the gut microbiota were associated with profound changes in host metabolite concentrations. Further, short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, were detected in cecal contents and serum by gas chromatography-mass spectrometry (GC-MS). We observed that the concentrations of these three SCFAs were all lower in the infected groups than in the untreated controls. Lastly, oral supplementation with these three SCFAs reduced susceptibility to K. pneumoniae infections, as indicated by lower bacterial burdens in the lung and higher survival rates. Our data highlight the protective roles of gut microbiota and certain metabolites in K. pneumoniae-pneumonia and suggests that it is possible to intervene in this bacterial pneumonia by targeting the gut microbiota.

[CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells inhibits their phagocytosis to Klebsiella pneumoniae].

Objective To construct cell line RAW264.7 with stable knockout of GPR43 gene using CRISPR/Cas9 system, and explore the role and mechanism of GPR43 gene in Klebsiella pneumoniae infection. Methods Three pairs of small-guide RNA (sgRNA) targeting the GPR43 gene were designed and inserted into plasmid pLenticrisprV2. The recombinant plasmid pLenticrisprV2 containing sgRNA was packaged using a lentivirus packaging system. RAW264.7 cells were transfected with viruses, and monoclonal cells were screened using puromycin. The genomic DNA was extracted from the amplified monoclonal cells. The GPR43 gene-related sequences were sequenced and compared with the wild-type GPR43 gene to confirm the cell line with successful knockout (GPR43-/- RAW264.7 cells). The expression of GPR43 protein was detected by Western blotting. After GPR43-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the changes in the expression of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) in the cells were detected using real-time quantitative PCR. Additionally, the phagocytic capacity of RAW264.7 cells after GPR43 knockout was observed. Results Western blotting confirmed that GPR43 protein was not expressed in the selected monoclonal cells, and DNA sequencing showed that 34 bases were missing at the insertion site of sgRNA, which proved that GPR43 gene was successfully knocked out. After GPR43-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the levels of IL-1ß, IL-6 and TNF-α expression in the cells were all lower than those in the control group, and the phagocytic capacity of GPR43-/- RAW264.7 cells to Klebsiella pneumoniae decreased. Conclusion CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells can inhibit their phagocytosis for Klebsiella pneumoniae and production of inflammatory cytokines.