| Klebsiella pneumoniae is one of the three largest conditional pathogenic bacteria in hospital, which belongs to Gram negative bacteria. Klebsiella pneumonia can cause a variety of antibiotics failure, leading to multiple-drug resistance, even pan-drug resistance. It induces serious infections and respiratory diseases, some severe cases will be life-threatening. Due to broad spread and pathogenic characteristics, Klebsiella pneumonia is also known as "superbugs". Besides the hospital-acquired Klebsiella pneumonia strains (HAPs), some community-acquired Klebsiella pneumonia strains (CAPs) appear constantly in recent years also can cause respiratory disease, which is worth attention as well.We studied HAPs and CAPs systematically by combining Sanger sequencing, the next generation sequencing and the SMRT sequencing. We collected 338 HAP strains from 7 areas (Beijing, Fujian, Henan, and Hebei etc.); we sequenced the MLST tags of each strain and detected the MIC values accordingly. We had a correlation analysis between SNPs in MLST and MICs of 338 HAPs. We have found two genes of each containing a SNP locus associated with 14 kinds of antibiotic resistance; also we found another two genes of each contaiing a SNP locus associated with 13 kinds of antibiotic resistance.We compared systematically the difference between HAPs and CAPs on gene type and copy number by using the next generation sequencing, especially in efflux pump genes, and found some group specific genes between HAPs and CAPs. In phelogenetic tree analysis, we found HAPs and CAPs were mixed in the topological structure, which means HAPs and CAPs were the same species under diferent environment pressures. We constructed the core and dispensable gene dataset of HAPs and CAPs by using pan-genome methods. The pan genome analysis shows that CAP core gene dataset is more than HAP core gene dataset on family number (CAP 2198 vs HAP 1752). The reason may due to environment pressure drive the core gene family number gain and loss. In dispensable gene aspect, HAPs and CAPs has each own specific gene family, HAP strains are mainly related to metabolism, such as Galactosyl transferase, hydrolytic enzymes, and DNA primers enzyme, etc, and CAP strains are associated with environmental adaptation, for instance integrase, outer membrane proteins, and glucose transferase, etc.We selected two HAPs for genome fined map construction from above mentioned strains by using SMRT sequencing. After genome assembly and annotation, we acquired a complete plasmid genome of Klebsiella pneumonia strain H11 and a complete chromosome genome of Klebsiella pneumonia of strain H39. The bioinformatics analysis showed that the strain H11 plasmid genome carries multi-resistant genes:such as blaTEM-1, blaSHV-12, sull, qacE delta 1, ereA, arr2, aac3, etc. Meahwhile, we discovered three heavy metal resistance gene clusters (tellurium, lead and mercury). H11 has two independent toxin-antitoxin systems (HipBA and RelBE), which is not common in Klebsiella pneumonia strains. The strain H39 shows more unqiue featues. Although H39 was isolated from hostipal; it has a close relationship with an endophytic Klebsiella variicola strain 342 on evolutionary status. In singe gene (parC) phelogenetic tree analysis, H39 was clustered into group III, this kind of Klebsiella pneumoniae is usually related to the endophytic Klebsiella variicola strains. The bioinformatics analysis showed that H39 earned 46 genomic islands (GI) and 48 resistant genes. The comparative genomic analysis shows H39 has the unbroken nitrogen fixation gene cluster and this structure is special for endophytic Klebsiella variicola strains. It implies H39 may be a special strain which can infect animals and plants at the same time. |
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