Role of outer membrane impermeability in Pseudomonas aeruginosa resistance to liptin d7

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that is intrinsically resistant to hydrophobic molecules, yet is susceptive to chemical sensitization by low concentrations of the biocide triclosan using the outer membrane permeabilizer compound 48/80. In order to obtain a better understanding of these properties in P. aeruginosa, it was decided to examine disparate hydrophobic substances thought to also possess antibacterial potential. Researchers working with Dr. Dennis Burns at Wichita State University have recently synthesized and characterized a novel class of picket porphyrins (liptins) and showed them to have antibacterial properties in previous studies.METHODS: Minimal inhibitory concentrations (MICs) were determined for a model liptin molecule (d7) using a conventional macro-broth dilution bioassay. Pasteurella multocida was included as a reference organism because of its permeable outer membrane. The next steps would have been to repeat the analysis in the presence of outer membrane permeabilizer compound 48/80, as well as to perform disc agar diffusion assays to eliminate possible bactericidal interference by the solvent.RESULTS: The liptin d7 did not readily dissolve in absolute ethanol, thereby resulting in a turbid suspension at concentrations necessary for this study. P. aeruginosa and P. multocida were both found to be resistant to liptin d7 with MICs of 32 and 64 ug/mL, respectively.CONCLUSION: Because the MICs were so high, especially with respect to P. multocida which was expected to be susceptible, both organisms were deemed liptin d7 resistant. We were subsequently informed by our collaborators that the liptin derivative sent to us may have been incorrectly labeled and was in fact a less soluble and less inhibitory derivative than the intended liptin d7. This halted further study of the compound due to an inability to obtain the more soluble active form to perform the analyses

Characterization of growth and antibiotic resistance in Burkholderia cepacia complex organisms

Introduction: Isolates are opportunistic pathogens within the Burkholderia cepacia Complex (Bcc). One key characteristic is that they are gram-negative, thereby making them generally resistant to hydrophobic molecules.Hypothesis: Exclusionary properties of the gram-negative outer membrane for hydrophobic substances are at least partially responsible in cases where clinical isolates of Bcc species are intrinsically resistant to hydrophobic antibacterial agents.Study Design: This hypothesis will be tested using turbidimetric growth and disk agar diffusion and bioassays.Methods: Test samples were cultivated in a shaking incubator for six hours at 180 rpm and 37 degrees Celsius to determine their respective growth rates. Spectrophotometric readings were taken every half hour to measure optical density at 620 nm wavelength. Test samples were also grown to 0.1 optical density and then streaked onto a Petri dish containing Mueller Hinton agar. Eight different antibiotic discs were dispensed onto the seeded agar surfaces and compounds were allowed to diffuse at three degrees Celsius for one hour, and then samples were incubated for 24 hours at 37 degree Celsius.Results: All Bcc isolates exhibited similar sigmoid growth curves and yielded similar amounts of biomass. Overall, they were slightly susceptible to triclosan and rifampin, and moderately to very susceptible to clindamycin and novobiocin.Conclusion: The Bcc isolates were pure cultures and exhibited similar in vitro growth characteristics. Their similar patterns of resistance and susceptibility to hydrophobic antibacterial agents suggests phenotypic outer membrane exclusion properties for hydrophobic molecules is largely conserved among the Burkholderia species examined

Extracellular Compounds Having Antibacterial Properties Produced by the Entomopathogenic Fungus Beauveria Bassiana

Microbiolog

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

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