Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Lytic Effect of Terpeneless Cold Pressed Valencia Orange Oil on Methicillin-Resistant Staphylococcus aureus

ABSTRACT: Staphylococcus aureus has been recognized as an important cause of human disease for more than 100 years. Resistance to multiple classes of antibiotics is becoming an increasingly difficult problem in the management of methicillin-resistant S. aureus (MRSA) and vancomycin-intermediate resistant S. aureus (VISA) infections. One approach to the MRSA and VISA problem, involves the discovery and development of new natural antimicrobials. The antimicrobial properties of essential oils of plant origin have been recognized for many years. In this study 0.1% of commercial cold pressed terpeneless Valencia orange oil (CPV) showed inhibitory and lytic activity against MRSA and VISA. To identify the mechanisms of action of CPV genomic response of CPV treated MRSA was analyzed by transcriptional profiling. Results showed alteration in the expression of cell wall peptidoglycan biosynthesis associated genes in the CPV treated cells. Transmission electron microscopic observation of CPV treated MRSA cells exhibited cell wall damage and cell lysis. Overall results of this study suggest that CPV may be a potential anti-staphylococcal agent for MRSA. Overnight grown S. aureus COL was inoculated in TSB medium (20 ml) in a 50 ml Erlenmeyer flask and incubated at 37°C, with shaking at 200 rpm. Growth was measured at regular intervals at 600 nm until OD reached approximately 0.4. Based on the GIC study ½ x MIC concentration of EO was added for 15 min of challenge. Control cultures were not challenged with EO and were also incubated for 15 min. Total bacterial RNA was isolated as previously described and the RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 6 (NIAID's Pathogen Functional Genomics Resource Center). Hybridization signals were scanned using an Axon4000B scanner (Molecular Devices, Sunnyvale, CA ) with Acuity 6.0 software and scans were saved as TIFF image. Scans were analyzed using TIGR-Spotfinder (www.tigr.org/software/) software and the local background was subsequently subtracted. The data set was normalized by applying the LOWESS algorithm using TIGR-MIDAS (www.tigr.org/software/) software. The normalized log2 ratio of test/reference signal for each spot was recorded. Genes with less than three data points were considered unreliable, and their data points were discarded. The averaged log2 ratios for each remaining gene on the 6 replicate slides were ultimately calculated. Significant changes of gene expression were identified with SAM (significance analysis of microarrays; www.tat.stanford.edu/~tibs/SAM/index.html. Contents of raw data files: Channel A = Cy3 dye Channel B = Cy5 dye

ORGANISM(S): Staphylococcus aureus

SUBMITTER: Arunachalam Muthaiyan

PROVIDER: E-GEOD-33465 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 Âµg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 Âµg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus. Growth conditions for microarray analysis- For transcriptional profiling, an overnight-grown COL culture was diluted (2% vol/vol) to 20 ml TSB medium in a 50-ml Erlenmeyer flask and grown at 30Â°C with shaking at 200 rpm. For the sensitization experiment, COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. Total bacterial RNA was isolated as previously described from S. aureus COL (30Â°C, 200 rpm). The RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 7 (NIAID's Pathogen Functional Genomics Resource Center). Hybridization signals were scanned using an Axon4000B scanner (Molecular Devices, Sunnyvale, CA ) with Acuity 4.0 software and scans were saved as TIFF image. Scans were analyzed using TIGR-Spotfinder (www.tigr.org/software/) software and the local background was subsequently subtracted. The data set was normalized by applying the LOWESS algorithm using TIGR-MIDAS (www.tigr.org/software/) software. The normalized log2 ratio of test/reference signal for each spot was recorded. Genes with less than three data points were considered unreliable, and their data points were discarded. The averaged log2 ratio for each remaining gene on the four replicate slides was ultimately calculated. Significant changes of gene expression were identified with SAM (significance analysis of microarrays; www.tat.stanford.edu/~tibs/SAM/index.html. Genes analyzed using these programs were further sorted and grouped based on their function using our in-house software Staphylococcus aureus Gene Sorter (SAGS). Several controls were employed to minimize the technical and biological variations and to ensure that the data obtained were of good quality. First, each ORF was present in triplicate on the array. Second, each RNA preparation was used to make probes for at least two separate arrays for which the incorporated dye was reversed. Contents of raw data files: Channel A = Cy3 dye Channel B = Cy5 dye File names ending with S1,S3 = Cy3 untreated, Cy5 treated File names ending with S2,S4 = Cy5 untreated, Cy3 treated

Project description:The precise mechanism and effects of antibiotics in host gene expression and immunomodulation in MRSA infection is unknown. Using a well characterized Methicillin Resistant Staphylococcus aureus (MRSA) isolate USA300 in a murine model of infection, we determined that linezolid and vancomycin induced differential production of bacterial toxins and host cytokines, differences in host gene expression, and differences in immunomodulators during MRSA bloodstream infection. A total of 35 A/J mice, categorized into seven groups (no infection; no infection with linezolid; no infection with vancomycin; 2 hour post-infection (hpi) S. aureus; 24 hpi S. aureus; 24 hpi S. aureus with linezolid; and 24 hpi S. aureus with vancomycin), were used in this study. Mice were injected with USA300 (6 x 106 CFU/g via i.p. route), then intravenously treated with linezolid (25 mg/kg) or vancomycin (25 mg/kg) at 2 hpi. Control and S. aureus infected mice were euthanized at each time point (2 h or 24h) following injection. Whole blood RNA was used for microarray; three cytokines and two S. aureus toxins [PantonValentine Leukocidin (PVL) and alpha hemolysin] were quantified in mouse serum by ELISA. S. aureus CFUs were significantly reduced in blood and kidney after linezolid or vancomycin treatment in S. aureus-infected mice. In vivo IL-1M-NM-2 in mouse serum was significantly reduced in both linezolid (p=0.001) and vancomycin (p=0.006) treated mice compared to untreated ones. IL-6 was significantly reduced only in linezolid treated (p<0.001) but not in vancomycin treated mice. However, another proinflammatory cytokine, TNF-M-NM-1, did not exhibit altered levels in either linezolid or vancomycin treated mice (p=0.3 and p=0.51 respectively). In vivo level of bacterial toxin, Panton-Valentine leukocidin, in mouse serum was significantly reduced only in linezolid treated mice (p=0.02) but not in vancomycin treated mice. There was no significant effect of either treatment in in vivo level of alpha hemolysin production. Unsupervised hierarchical clustering using the gene expression data from 35 microarrays revealed distinct clustering based on infection status and treatment group. Study of the antibiotic-specific difference in gene expression identified the number of genes uniquely expressed in response to S. aureus infection, infection with linezolid treatment, and infection with vancomycin treatment. Pathway associations study for the differentially expressed genes in each comparison group (Control vs. 24 h S. aureus infection, 24 h S. aureus infection vs. 24 h S. aureus linezolid, and 24 h S. aureus infection vs. 24 h S. aureus vancomycin) in mice using Kyoto Encyclopedia of Genes and Genomes (KEGG) identified toll-like receptor signaling pathway to be common to every comparison groups studied. Glycerolipid metabolism pathway was uniquely associated only with linezolid treatment comparison group. The findings of this study provide the evidence that protein synthesis inhibitor like linezolid does a better job in treating MRSA sepsis compared to cell wall acting antibiotics like vancomycin. To identify differences in host gene expression in a murine sepsis model treated with a) linezolid and b) vancomycin, we used whole blood gene expression (RNA) signatures from A/J inbred mice infected with USA 300 MRSA to evaluate differences in host gene expression among mice treated with linezolid and vancomycin. We used 5 RNA samples from MRSA-infected, linezolid- or vancomycin-treated mice. A total of 7 experimental groups have been employed: 1) Uninfected control group: (negative controls). 2) Uninfected, linezolid-treated group: Uninfected, linezolid-treated mice. 3) Uninfected vancomycin-treated group: Uninfected, vancomycin-treated mice. 4) Infected control group (positive control 2 h) MRSA-infected, untreated mice. 5) Infected control group (positive control 24 h): MRSA-infected, untreated mice. 6) Infected linezolid group: MRSA-infected, linezolid-treated mice. 7) Infected vancomycin group: MRSA-infected, vancomycin-treated mice.

Dataset Information

Lytic Effect of Terpeneless Cold Pressed Valencia Orange Oil on Methicillin-Resistant Staphylococcus aureus

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure