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A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

ABSTRACT: Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.

SUBMITTER: Selin C

PROVIDER: S-EPMC4460083 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

Selin Carrie C Stietz Maria S MS Blanchard Jan E JE Gehrke Sebastian S SS Bernard Sylvain S Hall Dennis G DG Brown Eric D ED Cardona Silvia T ST

PloS one 20150608 6

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well ...[more]

PMID: 26053039

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Project description:Burkholderia cenocepacia J2315 is a member of the B. cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B. cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, ΔncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B. cenocepacia J2315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in ΔncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B. cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B. cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to "cepacia syndrome," a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B. cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B. cenocepacia that are relevant for infection.

Project description:Antibiotics are considered to be the first line of treatment for mild to moderately severe Clostridium difficile infection (CDI) in humans. However, antibiotics are also risk factors for CDI as they decrease colonization resistance against C. difficile by altering the gut microbiota and metabolome. Finding compounds that selectively inhibit different stages of the C. difficile life cycle, while sparing the indigenous gut microbiota is important for the development of alternatives to standard antibiotic treatment. 2-aminoimidazole (2-AI) molecules are known to disrupt bacterial protection mechanisms in antibiotic resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, but are yet to be evaluated against C. difficile. A comprehensive small molecule-screening pipeline was developed to investigate how novel small molecules affect different stages of the C. difficile life cycle (growth, toxin, and sporulation) in vitro, and a library of commensal bacteria that are associated with colonization resistance against C. difficile. The initial screening tested the efficacy of eleven 2-AI molecules (compound 1 through 11) against C. difficile R20291 compared to a vancomycin (2 ?g/ml) control. Molecules were selected for their ability to inhibit C. difficile growth, toxin activity, and sporulation. Further testing included growth inhibition of other C. difficile strains (CD196, M68, CF5, 630, BI9, M120) belonging to distinct PCR ribotypes, and a commensal panel (Bacteroides fragilis, B. thetaiotaomicron, C. scindens, C. hylemonae, Lactobacillus acidophilus, L. gasseri, Escherichia coli, B. longum subsp. infantis). Three molecules compound 1 and 2, and 3 were microbicidal, whereas compounds 4, 7, 9, and 11 inhibited toxin activity without affecting the growth of C. difficile strains and the commensal microbiota. The antimicrobial and anti-toxin effects of 2-AI molecules need to be further characterized for mode of action and validated in a mouse model of CDI.

Project description:Reduced antimicrobial susceptibility due to resistance and tolerance has become a serious threat to human health. An approach to overcome this reduced susceptibility is the use of antibiotic adjuvants, also known as potentiators. These are compounds that have little or no antibacterial effect on their own but increase the susceptibility of bacterial cells towards antimicrobial agents. Baicalin hydrate, previously described as a quorum sensing inhibitor, is such a potentiator that increases the susceptibility of Burkholderia cenocepacia J2315 biofilms towards tobramycin. The goal of the present study is to elucidate the molecular mechanisms behind the potentiating activity of baicalin hydrate and related flavonoids. We first determined the effect of multiple flavonoids on susceptibility of B. cenocepacia J2315 towards tobramycin. Increased antibiotic susceptibility was most pronounced in combination with apigenin 7-O-glucoside and baicalin hydrate. For baicalin hydrate, also other B. cepacia complex strains and other antibiotics were tested. The potentiating effect was only observed for aminoglycosides and was both strain- and aminoglycoside-dependent. Subsequently, gene expression was compared between baicalin hydrate treated and untreated cells, in the presence and absence of tobramycin. This revealed that baicalin hydrate affected cellular respiration, resulting in increased reactive oxygen species production in the presence of tobramycin. We subsequently showed that baicalin hydrate has an impact on oxidative stress via several pathways including oxidative phosphorylation, glucarate metabolism and by modulating biosynthesis of putrescine. Furthermore, our data strongly suggest that the influence of baicalin hydrate on oxidative stress is unrelated to quorum sensing. Our data indicate that the potentiating effect of baicalin hydrate is due to modulating the oxidative stress response, which in turn leads to increased tobramycin-mediated killing.

Project description:Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia, focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth.

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A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

Publications

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

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