Antibacterial properties of novel Eumelanin-Inspired Phenylene Indolyne derivatives

BACKGROUND: The eumelanin core represents a novel compound having the intrinsic ability to act as scaffolding for functional groups which may possess antibacterial properties. The purpose of this study was to investigate the antibacterial potential of eumelanin-inspired phenylene indolyne (EIPE) derivatives EIPE-1 and EIPE-HCl which are hydrophobic and hydrophilic, respectively.METHODS: A standardized disk agar diffusion bioassay was employed to determine the susceptibility and resistance levels of 12 gram-positive and 13 gram-negative bacteria to nonpolar and polar EIPE derivatives. The bioassay was performed by dissolving the compounds in dimethyl sulfoxide and impregnating filter paper disks which were placed onto Mueller Hinton agar plates spread inoculated in a standardized manner to obtain even cell lawns after incubation for 18±1 hours at 37°C. Zones of growth inhibition were measured with the aid of electronic calipers.RESULTS: Five strains of Staphylococcus aureus, plus Bacillus subtilis and Staphylococcus epidermidis were all found to be susceptible to the hydrophobic derivative EIPE-1, while other gram-positive and all gram-negative organisms exhibited resistant phenotypes at potencies tested. The more polar EIPE-HCl derivative failed to inhibit growth of any of the organisms examined, regardless of gram reactivity.CONCLUSION: Hydrophobic EIPE derivative EIPE-1 clearly possesses a gram-positive antibacterial spectrum, although only certain organisms are susceptible at the potencies employed for this study. The susceptibility of two methicillin-resistant S. aureus strains (SFL 8 and SFL 64) to EIPE-1 suggests that its mechanism of action does not involve the penicillin-binding proteins of peptidoglycan biosynthesis targeted by mainstream B-lactam antibiotics. The uniform resistance of 13 phylogenetically disparate gram-negative bacteria supports the notion that intrinsic outer membrane exclusion properties may play a role in the mechanism underlying their phenotypic resistance to the molecule. The more polar EIPE-HCl possesses no antibacterial properties at the potencies examined here. Future work will include performing minimal inhibitory concentration bioassays to quantitatively describe susceptibly in selected gram-positive bacteria. In addition, batch culture growth kinetics assays will be crucial to learning the cellular and molecular mechanisms responsible for susceptibility and resistance to EIPE-1

Effects of gram-negative bacterial outer membrane permeabilization on Serratia marcescens' gene expression

Our laboratory has focused on understanding the effects of outer membrane permeabilizer compound 48/80 on the intrinsic resistance of gram-negative bacteria to hydrophobic antibacterial agents such as the biocide triclosan. Previously obtained RNAseq data were analyzed to identify gene expression changes potentiated by compound 48/80 in the opportunistic pathogen Serratia marcescens. These data indicated greatly upregulated expression of three genes, each involved in repair of outer membrane damage by other antimicrobial agents. The objective of the present study was to clarify the bacterial response to compound 48/80 treatment using RT-qPCR to follow gene expression, with the ultimate goal of establishing a proposed mechanism of action for compound 48/80-induced outer membrane permeability. Previous work indicated that S. marcescens is one of the few species of bacteria intrinsically resistant to triclosan, and that compound 48/80 induces transient sensitization to triclosan. The RNAseq analyses revealed a 50-fold increase in expression of slyB, phoP, and phoQ subsequent to compound 48/80 administration and qPCR primers were created in order to further investigate their regulation. The Bacterial and Viral Bioinformatics Resource Center (BV-BRC) analysis tools confirmed the upregulation of the aforementioned genes and provided Fragments Per Kilobase Million Mapped Reads (FPKM) to enable selection of an appropriate qPCR housekeeping gene, yfiR. RNA has been prepared from expression time-courses in preparation for the RTqPCR process, in which qPCR primers for slyB, phoQ, phoP and yfiR will be used to measure expression changes observed over time

Contribution of Choline-Binding Proteins to Cell Surface Properties of Streptococcus pneumoniae

Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions

Draft Genome Sequence of Enterobacter cloacae Strain JD6301

Enterobacter cloacae strain JD6301 was isolated from a mixed culture with wastewater collected from a municipal treatment facility and oleaginous microorganisms. A draft genome sequence of this organism indicates that it has a genome size of 4,772,910 bp, an average G+C content of 53%, and 4,509 protein-coding genes

Growth kinetics of <i>P</i>. <i>aeruginosa</i> PAO1 in the presence of novobiocin (10.0 μg/mL) and the indicated outer membrane permeabilizer.

Each value represents the mean of at least three independent determinations. Symbols: (●) control, (○) novobiocin, (▼) permeabilizer, (△) novobiocin plus permeabilizer.</p

Growth kinetics of <i>B</i>. <i>multivorans</i> CGD2 in the presence of novobiocin (1.0 μg/ml) and the indicated outer membrane permeabilizer.

Each value represents the mean of at least three independent determinations. Symbols: (●) control, (▼) novobiocin, (■) permeabilizer, (♦) novobiocin plus permeabilizer. (TIF)</p

Susceptibility to outer membrane impermeant antibacterial agents and polymyxin B.

Susceptibility to outer membrane impermeant antibacterial agents and polymyxin B.</p

Growth kinetics of <i>P</i>. <i>aeruginosa</i> PAO1 in the presence of triclosan (2.0 μg/mL) and the indicated outer membrane permeabilizer.

Each value represents the mean of at least three independent determinations. Symbols: (●) control, (○) triclosan, (▼) permeabilizer, (△) triclosan plus permeabilizer.</p

Growth kinetics of <i>B</i>. <i>multivorans</i> ATCC BAA-247 (type strain) in the presence of triclosan (1.0 μg/mL) and the indicated outer membrane permeabilizer.

Each value represents the mean of at least three independent determinations. Symbols: (●) control, (○) triclosan, (▼) permeabilizer, (△) triclosan plus permeabilizer.</p