Project description:Objective: It is unclear whether the host response of gram-positive sepsis differs from gram-negative sepsis at a transcriptome level. Using microarray technology, we compared the gene-expression profiles of gram-positive sepsis and gram-negative sepsis in critically ill patients. Design: A prospective cross-sectional study. Setting: A 20-bed general intensive care unit of a tertiary referral hospital. Patients: Seventy-two patients admitted to the intensive care unit. Interventions: Intravenous blood was collected for leukocyte separation and RNA extraction. Microarray experiements were then performed examing the expression level of 19,232 genes in each sample. Measurements and Main Results: There was no difference in the expression profile between gram-positive and gram-negative sepsis. The finding remained unchanged even when genes with lower expression level were included or after statistical stringency was lowered. There were, however, ninety-four genes differentially expressed between sepsis and control patients. These genes included those involved in immune regulation, inflammation and mitochondrial function. Hierarchical cluster analysis confirmed that the difference in gene expression profile existed between sepsis and control patients, but not between gram-positive and gram-negative patients. Conclusion: Gram-positive and gram-negative sepsis share a common host response at a transcriptome level. These findings support the hypothesis that the septic response is non-specific and is designed to provide a more general response that can be elicited by a wide range of different micro-organisms. The study included seventy-two critically ill patients admitted to the intensive care unit (ICU) of Nepean Hospital, Sydney, Australia. Of these, fifty-five patients were diagnosed to have sepsis, as confirmed by microbiological culture. The remaining seventeen patients did not have sepsis and were therefore used as controls. The study was approved by the hospital ethics committee and informed consent was obtained from all patients or their relatives. Patient Samples. Whole blood was taken from each patient on admission to ICU. Neutrophils were separated from whole blood using density-gradient separation with Ficoll-PaqueP P(Amersham). Subsequent neutrophil RNA extraction was performed using guanidinium thiocyanate (Ambion). Microarray Experiment. The neutrophil RNA was converted to cDNA, fluorescently labeled and hybridized to its complimentary sequences on the microarray (Invitrogen). The fluorescent signals on each micrroarray were captured using the GenePix 4000B laser scanner (Axon Instruments). Expression level of each gene was represented by the intensity of its fluorescent signal. Data Extraction. All signal intensity values were processed using background-subtraction method. Prior to analysis, all values were log-transformed and normalized by fitting a print-tip group Lowess curve. Normalization minimizes bias due to dye chemistry, signal intensity or location of a gene on the array. It ensures the detection of genes that are truly differentially expressed, instead of those caused by experimental artifacts or variation in the hybridization process. After normalization, genes that had more than 50% of data missing were removed. We then selected genes that had at least 80% of the data showing two-fold changes from the geneâs median values. After filtering, 1617 genes were available for further analysis.

Project description:We profiled the expression of circulating microRNAs (miRNAs) in mice exposed to gram-positive and gram-negative bacteria using Illumina small RNA deep sequencing. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. Male C57BL/6 mice (age, 10–12 weeks; weight, 30–35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The mice were anesthetized by intraperitoneal injection of an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine. The anesthetized mice were restrained in a supine position on a heated pad to maintain body temperature at 37°C. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) purchased from Caliper (Caliper, USA) were used to induce bacterial infection in the mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). To create mixed gram-negative and gram-positive bacterial infection, 1 × 108 Xen14 bacteria and 1 × 108 Xen29 bacteria/100 μL of PBS were used for wound contamination. Three animal models were used to create bacterial infection routes: subcutaneous injection (hereafter referred to as (I)), cut wound (hereafter referred to as (C)), and skin grafting (hereafter referred to as (S)). In the (I) model, E. coli and/or S. aureus suspensions were injected subcutaneously into the backs of the mice using an Fr. 25 needle. In the (C) model, a 1 cm incision wound was created in the midline of the back, smeared with E. coli and/or S. aureus suspension, and the wound was closed directly with a 4-0 nylon suture. In the (S) model, a 1×1 cm rectangular full- thickness skin graft was lifted from the backs of the mice, E. coli and/or S. aureus suspensions were spread over the wound bed, and the skin graft was reattached and closed with a 4-0 nylon suture. An additional group of animals in each of these three models was inoculated with PBS to serve as a negative control. Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer.

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Identification of Gram-positive bacteria using BC-GP assay

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