Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Beta- lactam antibiotics stimulate biofilm formation in non-typeable Haemophilus influenzae by up-regulating carbohydrate metabolism

ABSTRACT: Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10M-BM-5g/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. 12 samples

ORGANISM(S): Haemophilus influenzae

SUBMITTER: Charles Wang

PROVIDER: E-GEOD-58213 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:We investigated gene expression levels in Heliconius erato butterflies with divergent wing patterns across a 656KB genomic interval linked to the red color pattern wing polymorphism. This included comparison of expression between two H. erato color pattern populations (H. e. petiverana and a H.e. etylus x H. himera hybrid) across three sections of the forewing that differed in pigmentation (the basal, mid, and distal wing sections) and five different stages of pupal development (Day 1, 3, 5 pupae and ommochrome and melanin pigmentation stages). These results allowed us to determine whether certain genes in this interval were differentially expressed between the wing pattern elements, and, therefore, potentially responsible for adaptive color pattern variation in these butterflies. Forewings from a total of 29 individuals, covering three biological replicates of five developmental time points for each of the two H. erato distinct phenotypes were dissected, with the only exception being that there were only two replicates of the day 1 hybrid phenotype. Individuals were reared at 25˚C and dissected at the following stages: a) day 1 = 12 hr after pupation; b) day 3 = 60 hr after pupation; c) day 5 = 108 hr after pupation; d) early ommochrome = ~ 156 hr after pupation when red scales in forewing partially mature, showing a pale orange color; and e) early melanin = ~ 180 hr after pupation melanic scales begin to turn black and are present primarily at the center of the wing. Using wing veins as landmarks, each forewing was cut into three sections corresponding to the color pattern boundaries: basal (F1), middle (F2), and apical (F3). A eight custom-designed Roche NimbleGen 12x135K format microarrays with probes spanning a 656,307bp genomic region (Roche NimbleGen Inc., Madison, Wisconsin, United States) were used to hybridize double stranded cDNA from 87 tissue samples. Repetitive sequence elements found more than five times across all currently available H. erato genomic sequences, including the probed region as well as additional genomic BAC sequences, were masked from the tiling region. The remaining unmasked non-repetitive genomic sequences were tiled using 60 bp probes staggered every 13 bp on average, with slight modifications to ensure probe quality, for a total of 48,547 probes. Microarry design and printing was performed by Roche NimbleGen. cDNA labeling, hybridization, and array scanning was performed by the City of Hope Microarray Facility (Duarte, California, United States). In addition to the probes from the red color pattern intervals, the arrays also include 40,763 probes across two other genomic intervals not addressed in this study, 45,046 probes representing 12450 transcripts from a recent transcriptome assembly at 1-6X coverage, and 3248 random probes. Results from the transcriptome and other color pattern intervals will be published separately, however, we analyzed all probes together for array normalization and quality control.

Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea. Four sample groups of N. europaea cells were used in this study, with biological triplicates of each group. Groups were: Control (untreated) biofilms, phenol-exposed biofilms, toluene-exposed biofilms, and exponentially growing suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors containing 4 independent growth channels and subject to 2 hour inhibition tests. During each experiment, 2 biofilm channels served as control with no inhibitor present and the other 2 biofilm channels were exposed to either 60 uM phenol or 100 uM toluene. Nitrite production was monitored throughout the experiment, and the given concentrations of phenol and toluene resulted in 50% inhibition of ammonia oxidation by the biofilms. Suspended cells were grown in batch reactors. Three 4-plex NimbleGen microarray chips were used, and each chip contained one sample from each experimental group. QC of samples was determined by spectrophotometric methods and using Agilent bioanalyzer traces to determine purity and integrity of RNA and cDNA. A sample tracking report was used to verify the correct hybridization of each sample to the intended array.

Project description:Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptional responses of Pseudomonas aeruginosa growing in biofilms by using microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNAs for these housekeeping genes were low in cells at the bottom of the biofilms. Selective GFP labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNAs levels for genes regulated by the hypoxia induced regulator, Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-termanoxia. Transcripts for ribosomal proteins were primarily associated with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (rmf and PA4463) at the bottom of the biofilms. A P. aeruginosa M-bM-^HM-^Frmf strain had increased uptake of the membrane integrity stain, propidium iodide. Using selective GFP labeling and cell sorting, we showed that the dividing cells were more susceptible to tobramycin and ciprofloxacin than the dormant subpopulation. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. 52-hour Pseudomonas aeruginosa TSA colony biofilms were cryoembedded, thin sectioned, and laser dissected (LCM) to obtain samples from the top and bottom 50 M-BM-5m of the biofilms. 9 sections per biofilm were pooled. RNA was extracted with the RNeasy Micro kit, Turbo DNase treated, poly(A) tailed, and amplified using the Quantitect WTA kit. After clean up, the resulting product was fragmented and end labeled before hybridization.

Project description:Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes defcient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-defcient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function. [i] H3 ChIP-chip measurements of WT human cytomegalovirus (strain TB40E) following infection of MRC-5 cells. WT 8 hours postinfection, with corresponding mock IgG input control: 3 replicates; WT virus, 48 hours postinfection with corresponding mock IgG input contro: 3 replicates. [ii] MNase-chip measurements of WT and dlIE1 human cytomegalovirus nucleosomes following infection of MRC-5 cells. WT 8 hours postinfection, with corresponding sonicated DNA input control: 6 replicates; WT virus 48 hours postinfection, with corresponding sonicated DNA input control: 6 replicates; WT 96 hours postinfection, with corresponding sonicated DNA input control: 2 replicates; dlIE1 virus 8 hours postinfection, with corresponding sonicated DNA input control: 2 replicates; dlIE1 virus 96 hours postinfection, with corresponding sonicated DNA input control: 2 replicates. [iii] Total and nascent RNA measurements of WT and dlIE1 human cytomegalovirus transcripts following infection of MRC-5 cells. WT 8 hours postinfection: 2 replicates; WT virus 96 hours postinfection: 2 replicates; dlIE1 8 hours postinfection: 2 replicates; dlIE1 virus 96 hours postinfection: 2 replicates; sonicated DNA input control: 10 replicates.

Dataset Information

Beta- lactam antibiotics stimulate biofilm formation in non-typeable Haemophilus influenzae by up-regulating carbohydrate metabolism

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets