Further Insights into the Mode of Action of the Lipoglycopeptide Telavancin through Global Gene Expression Studies
ABSTRACT: Background: Telavancin is a novel semi-synthetic lipoglycopeptide derivative of vancomycin with a decylaminoethyl side chain that is active against Gram-positive bacteria including Staphylococcus aureus strains resistant to methicillin or vancomycin. This study describes transcriptome alterations in S. aureus strain ATCC29213 treated with telavancin for 15 min and 60 min in comparing with other agents treatment, including vancomycin, enduracidin, m-chlorophenylhydrazone. MHB cultures (biological replicates: N=3) were then grown to exponential phase (OD580=0.35) before the addition of telavancin(8 µg/ml final concentration), or vancomycin (10 /µg ml) or CCCP (2 µg/ ml ); or enduracidin (1 µg ml) and incubated for an additional 15 min , and 60 min before sampling. Dye-swapping was performed between samples.
Project description:Cultures of Y. pestis Kimberley53 virulent strain were exposed to different concentrations of ciprofloxacin (including 0.016 µg/ml representing the strains' MIC value) for various time periods. Total RNA samples were extructed and used for cRNA synthesis and labelling (using Cy3-CTP for the treated samples and Cy5-CTP for the non-treated sample in one biological experiment and dye swap for the 2nd replicate of independent biological experiment). 8 Labeled cRNA samples were hybridized to custom made Agilent 8x15K slide (each slide represent 1 time point). Goal: Identifing alterations in gene expression profile which correlates with the ciprofloxacin induced growth inhibition. Those altered mRNA transcrips will be used as a markers for the development of rapid molecular Antibiotic Susceptibility Test. Y. perstis exposed to 0.001, 0.016, 0.5 and 4 µg/ml ciprofloxacin, compared to growth control (non treated) sample, for 3 time periods (45, 90 and 120 min.). Two independent biological replicates were performed. Extracted total RNA were used for cRNA synthesis and labeling with Cy3/Cy5-CTP, hybridized to Agilent custom-made 8x15K slide format.
Project description:P388D1 murine macrophages were cultured in 85 mm tissue culture plates to about semi confluency. L. monocytogenes serotypes (1/2a EGD-e, 4a L99, 4b CLIP80459 and 4b F2365) were infected to the P388D1 cell monolayer at a MOI of 100 per eukaryotic cell. Infection was carried for 45 min and followed by addition of fresh medium containing 20 µg/ml gentamicin. The medium of the plates (containing 20 µg/ml gentamicin) infected with L. monocytogenes serotypes were replaced after 2 h post infection with fresh medium containing 50 µg/ml gentamicin. At each step the plates were washed extensively with 1x PBS. Incubation of the bacterial tissue culture plates was carried out in a humidified incubator for up to 4 h post infection.
Project description:Staphylococcus aureus is one of the most important pathogens in humans and animals, multiply resistant strains are increasingly widespread, new agents are needed for the treatment of S. aureus. Rhein, a natural plant product, has potential antimicrobial activity against Staphylococcus aureus. We employed Affymetrix Staphylococcus aureus GeneChipsTM arrays to investigate the global transcriptional profiling of Staphylococcus aureus ATCC25923 treated with rhein. Results provided insight into mechanisms involved in rhein - Staphylococcus aureus interactions. Keywords: rhein response Staphylococcus aureus cells were exposed for 45 minutes to rhein at concentration of 8 µg/ml (1/2× MIC), 6 samples including 3 control samples are analyzed.
Project description:We characterized the variation of M. tuberculosis transcriptional profile in response to inhibitory and subhinibitory concentrations of vancomycin. Overall design: Whole genome microarrays were employed to profile gene expression in M. tuberculosis H37Rv in response to inhibitory or subinibitory concentration of vancomycin. Inhibitory conditions were achieved by exposing bacteria to 400 µg/ml of vancomycin, a concentration exceeding 10 times the minimum inhibitory concentration (10X-MIC); in these conditions bacterial replication arrested rapidly without killing. Subhinibitory conditions were obtained growing the cells in the presence of 5 µg/ml of vancomycin (1/8-MIC) which allowed bacteria to grow with the same profile as untreated cells, although at a reduced rate. Following the addition of vancomycin at a concentartion 10X-MIC, the culture was incubated 1 h, or 4 hrs, and then analyzed to identify which genes were expressed differentially.
Project description:To understand the relationship between gene expression and capsule formation, H99 cells were cultured overnight at 37ºC in the following eight conditions: low iron medium with or without both 500 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM bathophenanthroline disulfonate (BPDS); phosphate-buffered saline (PBS) with or without 10% v/v fetal bovine serum; Dulbecco's Modified Eagle's Medium (from Sigma) in ambient air or 5% CO2; and Littman's medium with either 0.01 µg/ml or 1 µg/ml thiamine. All samples were hybridized against a common reference pool of total RNA. Three biological replicates were performed for each growth condition, with the exception of growth in DMEM.
Project description:30h of growth biofilms in microfermentors were exposed to caspofungin 0.5 µg/ml, caspofungin 5 µg/ml, fluconazole 80 µg/ml or amphotericin B 8 µg/ml. Control and antifungal-exposed biofilms were recovered at t= 0, 30, 60 and 120 min post exposure to antifungal.
Project description:This SuperSeries is composed of the following subset Series: GSE33907: Tannic acid (20 µg/ mL) treatment effect on transcriptome of Pseudomonas fluorescens Pf-5 GSE33908: Tannic acid (160 µg/ mL) treatment effect on transcriptome of Pseudomonas fluorescens Pf-5 Refer to individual Series
Project description:hVISA clinical strain Mu3 spontaneously generates VISA at an extremely high frequency (1x10-6 or greater) within its cell population. The VISA-converted mutant strains generally grow slower than their parent hVISA strain, but they usually form colonies on vancomycin-containing agar plates after 48 hours of incubation on population analysis. However, we noticed a curious group of VISA mutants whose colony formation is much more delayed and observed as discrete colonies only after 96 hours incubation. They have extremely prolonged lag-phase and doubling-times, but recorded vancomycin MICs of 8 to 24 mg/L when determined after 48 to 96 hours of incubation. We established strain Mu3-6RS having vancomycin MIC of 12 mg/L (at 72h) as a representative of the ‘slow VISA’ (sVISA) strains. Its cell wall was thickened, and autolytic activity was decreased as compared with those of the parent strain Mu3. Whole genome sequencing of Mu3-6RS revealed only one mutation, which substituted the 512th amino-acid sequence of RNA polymerase ß-subunit from Arg of Mu3 to Proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant loss of small colony morphology, cell-wall thickness and reduced autolytic activity. Sequencing of rpoB genes of the reverted strains revealed mutations affecting the 512th codon replacing the Proline of Mu3-6RS to Leucine, Serine, or Histidine. This study proposes sVISA as a discrete class of VISA phenotype, which can serve as a temporary shelter for S. aureus to survive relatively high concentrations of vancomycin. The sVISA spontaneously returns to hVISA when the threat of vancomycin passes by. The rpoB(R512P) mutation may be regarded as a ‘regulatory mutation’ switching on and off the reversible sVISA phenotype. 5 sample analysis using 60mer-oligo microarray, 3 rpoB mutant(R512P,R512L,R512S), 2 wild-type strain(Mu3, ΔIP)
Project description:To determine the response at the transcriptional level of S. coelicolor cells to treatment with 3 antibiotics that target distinct stages of cell wall biosynthesis, biological triplicate cultures of strain M600 were treated with sub-lethal concentrations (10 µg/ml) of vancomycin, bacitracin or moenomycin A. Samples were taken 0, 30, 60, 90 minutes after addition of the antibiotic. A negative control which received no drug treatment was also performed.
Project description:E. coli cultures were exposed to tellurite 0.5 µg/ml during 15 min. Total RNA was extracted and cDNA labeled probes were generated by reverse transcription using Alexa 555 and Alexa 647 fluorophores. These probes were used to hybridize genomic slides containing genomic arrays to determine global transcriptional changes. Two-conditions experiment, antibacterial vs. Untreated control cells. Biological replicates: 2 control, 2 toxicants exposed cells, independently grown and harvested. One replicate per array. Dye swap conditions.