Transcriptome analyses reveal antibiotic-resistant pathways in Klebsiella pneumonia
The increasing antibiotic resistance of Klebsiella pneumoniae poses a serious threat to global public health. To investigate the antibiotic resistance mechanism of Klebsiella pneumonia, we performed gene expression profiling analysis using RNA-seq data for clinical isolates of Klebsiella pneumonia, KPN16 and ATCC13883. Our results showed that mutant strain KPN16 is likely to act against the antibiotics through increased increased butanoate metabolism and lipopolysaccharide biosynthesis, and decreased transmembrane transport activity. Overall design: The K. pneumonia (ATCC13883 and KPN16) isolate was cultured to reach a turbidity of 0.05 at OD600. 2 mL bacterial culture was centrifuged at 12,000 rpm for 2 min for cell collection. Discard the supernatant and store the bacterial collection at -80 ?. In each group, triplicate samples were prepared under the same experimental conditions. RNA degradation and contamination was monitored on 1% agarose gels. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. mRNA was purified from total RNA using probes to remove rRNA. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand, and then second strand of cDNA were synthesized. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.