Project description:The increasing resistence and/or bacterial tolerance to bactericides, such as chlorhexidine, causes worrisome public health problems. Using transcriptomical and microbiological studies, we analysed the molecular mechanisms associated with the adaptation to chlorhexidine in two carbapenemase-producing strains of Klebsiella pneumoniae belonging ST258-KPC3 and ST846-OXA48.
Project description:To investigate the whole-genome gene expression difference between the wild-type and capsule deletion mutant in Klebsiella pneumoniae MGH 78578. The mutants analyzed in this study are further described in Huang T.W., Stapleton J.C., Chang H.Y., Tsai S.F., Palsson B.O., Charusanti P. Capsule removal via lambda-Red knockout system perturbs biofilm formation and fimbriae extression in Klesiella pneumoniae MGH 78578 (manuscript submission) A six chip study using total RNA recovered from three separate wild-type cultures and three separate cultures of a capsule deltion mutant of Klebsiella pneumoniae MGH 78578. The capsule gene cluster (KPN_02493 to KPN_02515) was entirely removed in the capsule deletion mutant. Each chip measures the expression level of 5,305 genes from Klebsiella pneumoniae MGH 78578 and the associated five plasmids (pKPN3, pKPN4, pKPN5, pKPN6 and pKPN7) with 50-mer oligo tiling array with 30-mer spacer.
Project description:The study aimed to characterize plasmids mediating carbepenem resistance in Klebsiella pneumoniae in Pretoria, South Africa. We analysed 56 K. pneumoniae isolates collected from academic hospital around Pretoria. Based on phenotypic and molecular results of these isolates, 6 representative isolates were chosen for further analysis using long reads sequencing platform. We observed multidrug resistant phenotype in all these isolates, including resistance to aminoglycosides, tetracycline, phenicol, fosfomycin, floroquinolones, and beta-lactams antibiotics. The blaOXA-48/181 and blaNDM-1/7 were manily the plasmid-mediated carbapenemases responsible for carbapenem resistance in the K. pneumoniae isolates in these academic hospitals. These carbapenemase genes were mainly associated with plasmid replicon groups IncF, IncL/M, IncA/C, and IncX3. This study showed plasmid-mediated carbapenemase spread of blaOXA and blaNDM genes mediated by conjugative plasmids in Pretoria hospitals.
Project description:To investigate the whole-genome gene expression difference between the wild-type and capsule deletion mutant in Klebsiella pneumoniae MGH 78578. The mutants analyzed in this study are further described in Huang T.W., Stapleton J.C., Chang H.Y., Tsai S.F., Palsson B.O., Charusanti P. Capsule removal via lambda-Red knockout system perturbs biofilm formation and fimbriae extression in Klesiella pneumoniae MGH 78578 (manuscript submission)
Project description:In this study, we implemented RNA sequencing to identify melatonin sensitive transcripts during culture maturation. This work demonstrates that majority of melatonin sensitive genes are growth stage specific. Melatonin caused differential gene expression of 81 transcripts during the exponential growth and 30 during early stationary phase. This indole molecule affects genes related to the biofilm formation, fimbria biogenesis, transcriptional regulators, carbohydrate transport and metabolism, phosphotransferase system (PTS), stress response, metal ion binding and transport. It is likely that differential expression of biofilm and fimbria related genes is responsible for differences in macrocolony area. Additionally, melatonin potentially helps Klebsiella aerogenes in host colonization.
Project description:Purpose: The goal of this study was to use RNA-seq to define the Klebsiella pneumoniae transcriptome recorded under 5 different experimental conditions, and to identify signature genes of each condition by comparing global transcriptional profiles. Methods: mRNA profiles were generated for Klebsiella pneumoniae CH1034 clinical isolate, in triplicate, by deep sequencing. Total RNAs were harvested from bacteria cultured at 37°C in M63B1 minimal media under different conditions: (i) planktonic aerobic condition at OD 620nm=0.250 (exponential growth-phase), (ii) overnight planktonic aerobic condition (stationnary growth-phase), (iii) biofilm in a flow-cell chamber after 7 hours of incubation (7-hours old biofilm), (iv) biofilm in a flow-cell chamber after 13 hours of incubation (13-hours old biofilm), (v) bacteria self-dispersed from biofilm recovered in the flow-cell effluent (biofilm-dispersed bacteria). Ribosomal RNAs were removed using the Bacteria Ribo-Zero Magnetic kit (Epicentre Biotechnologies). Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina), and 50bp single-reads were obtained by HiSeq 2000 (Illumina).The sequence reads that passed FastQC quality filters were mapped to the CH1034 genome using BurrowsâWheeler Aligner (BWA) (0.7.12-r1039 version). The transcript levels were determined using HTSeq-count (0.6.1p1 version) with union mode followed by DESeq (1.16.0 version) analysis. qRTâPCR validation was performed using SYBR Green assays. Results: We found that each condition has a specific transcriptional profile, and we identify 4 robust signature genes for each. Conclusion: Our study represents the first detailed analysis of K. pneumoniae transcriptomes under different experimental conditions generated by RNA-seq technology. The data reported here should permit the dissection of complex biologic functions involved in the transition between the sessile and planktonic modes of growth. Determination of the transcriptional profiling of Klebsiella pneumoniae under 5 different experimental conditions. mRNA profiles were generated for bacteria under exponential planktonic growth-phase, stationary planktonic growth-phase, 7 hours-old biofilm, 13 hours-old biofilm and biofilm-dispersed modes, each in three biological replicates, by deep sequencing using Illumina HiSeq
Project description:Bacterial infections of wounds are associated with poor healing and worse scarring. We sought to identify transcriptomic patterns associated with impaired healing of wounds infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) using a rabbit ear wound model. Wounds created on post-operative day (POD) 0 were infected on POD3, within the inflammatory phase of healing. On POD4 the infected wounds were harvested for microarray/transcriptome analysis. Other wounds with 24-hour infections were treated with topical antibiotic to promote biofilm formation and harvested on POD6 or POD12. On POD4 before antibiotic treatment, both wounds contained elevated transcripts that enriched predominantly into inflammation/infection-response pathways and functions characteristic of infiltrating leukocytes. But there were 5-fold more elevated transcripts in P.a.- than K.p.-infected wounds. Additionally, unique to P.a.-infected wounds, was a minor network of inflammation/infection-response molecules with predicted upstream regulation predominated by type I interferons. Also on POD4, Dnr-transcripts of both wounds were enriched into stress-response pathways such as EIF2 signaling. But there were 8-fold more Dnr-transcripts in P.a.- than K.p.-infected wounds, and many more of them enriched in the function, cell death, suggesting that resident dermal cells of P.a.-infected wounds failed to survive a more destructive P.a. infection. On POD6, following two days of antibiotic treatment, the biofilm-colonized wounds expressed magnitudes fewer inflammation and stress-response transcripts. However, a single regulatory network of P.a.-infected wounds was found to consist of Upr-transcripts enriching immune/infection-response functions predicted to be regulated by type I interferons, which was similar to the network unique to P.a.-infected wounds on POD4. On POD12, genes expressed by K.p.-infected wounds suggesting healing, while for P.a.-infected wounds they suggested stalled healing. The similarities and differences between the wound responses to these infections further define the molecular foundations of healing impaired by infections. Rabbit ear Wounds created on post-operative day (POD) 0 were infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) on POD3 and harvested on POD4 for RNA extraction. Other wounds with 24-hour infections were treated with topical antibiotic to promote biofilm formation and harvested on POD6 or POD12.