Bahareh Asadishad

Method for the direct observation and quantification of survival of bacteria attached to negatively or positively charged surfaces in an aqueous medium.

The risk of groundwater contamination by microbial pathogens is linked to their survival in the subsurface. Although there is a large body of literature on the inactivation behavior of suspended (planktonic) microorganisms, little is known about the inactivation of bacteria when attached to sand grain surfaces in groundwater aquifers. The main goal of this study was to develop a fluorescence-based experimental technique for evaluating the extent of inactivation over time of bacteria adhered onto a surface in an aqueous environment. Key features of the developed technique are as follows: (i) attached cells do not need to be removed from the surface of interest for quantification, (ii) bacterial inactivation can be examined in real-time for prolonged time periods, and (iii) the system remains undisturbed (i.e., the aqueous environment is unchanged) during the assay. A negatively or positively charged substrate (i.e., bare or coated glass slide) was mounted in a parallel-plate flow cell, bacteria were allowed to attach onto the substrate, and the loss of bacterial membrane integrity and respiratory activity were investigated as a function of time by fluorescence microscopy using Live/Dead BacLight and BacLight RedoxSensor CTC (5-cyano-2,3-ditolyl tetrazolium chloride) viability assays. These two different measures of bacterial inactivation result in comparable trends in bacterial inactivation, confirming the validity of the experimental technique. The results of this work show that the developed technique is sensitive enough to distinguish between the inactivation kinetics of different representative bacteria attached to either a negatively charged (bare glass) surface or a positively charged (coated glass) surface. Hence, the technique can be used to characterize bacterial inactivation kinetics when attached to environmentally relevant surfaces over a broad range of groundwater chemistries.

Alkaloids Modulate Motility, Biofilm Formation and Antibiotic Susceptibility of Uropathogenic Escherichia coli

Alkaloid-containing natural compounds have shown promise in the treatment of microbial infections. However, practical application of many of these compounds is pending a mechanistic understanding of their mode of action. We investigated the effect of two alkaloids, piperine (found in black pepper) and reserpine (found in Indian snakeroot), on the ability of the uropathogenic bacterium Escherichia coli CFT073 to colonize abiotic surfaces. Sub-inhibitory concentrations of both compounds (0.5 to 10 µg/mL) decreased bacterial swarming and swimming motilities and increased biofilm formation. qRT-PCR revealed a decrease in the expression of the flagellar gene (fliC) and motility genes (motA and motB) along with an increased expression of adhesin genes (fimA, papA, uvrY). Interestingly, piperine increased penetration of the antibiotics ciprofloxacin and azithromycin into E. coli CFT073 biofilms and consequently enhanced the ability of these antibiotics to disperse pre-established biofilms. The findings suggest that these alkaloids can potentially affect bacterial colonization by hampering bacterial motility and may aid in the treatment of infection by increasing antibiotic penetration in biofilms.

Induction of a State of Iron Limitation in Uropathogenic Escherichia coli CFT073 by Cranberry-Derived Proanthocyanidins as Revealed by Microarray Analysis

ABSTRACT Transcriptional profiles of uropathogenic Escherichia coli CFT073 exposed to cranberry-derived proanthocyanidins (PACs) were determined. Our results indicate that bacteria grown on media supplemented with PACs were iron deprived. To our knowledge, this is the first time that PACs have been shown to induce a state of iron limitation in this bacterium.

Cranberry impairs selected behaviors essential for virulence in Proteus mirabilis HI4320

Proteus mirabilis is an etiological agent of complicated urinary tract infections. North American cranberries (Vaccinium macrocarpon) have long been considered to have protective properties against urinary tract infections. This work reports the effects of cranberry powder (CP) on the motility of P. mirabilis HI4320 and its expression of flaA, flhD, and ureD. Our results show that swimming and swarming motilities and swarmer-cell differentiation were inhibited by CP. Additionally, transcription of the flagellin gene flaA and of flhD, the first gene of the flagellar master operon flhDC, decreased during exposure of P. mirabilis to various concentrations of CP. Moreover, using ureD-gfp, a fusion of the urease accessory gene ureD with gfp, we show that CP inhibits urease expression. Because we demonstrate that CP does not inhibit the growth of P. mirabilis, the observed effects are not attributable to toxicity. Taken together, our results demonstrate that CP hinders motility of P. mirabilis and reduces the expression of important virulence factors.

Effect of Dissolved Oxygen on Two Bacterial Pathogens Examined using ATR-FTIR Spectroscopy, Microelectrophoresis, and Potentiometric Titration

The effects of dissolved oxygen tension during bacterial growth and acclimation on the cell surface properties and biochemical composition of the bacterial pathogens Escherichia coli O157:H7 and Yersinia enterocolitica are characterized. Three experimental techniques are used in an effort to understand the influence of bacterial growth and acclimation conditions on cell surface charge and the composition of the bacterial cell: (i) electrophoretic mobility measurements; (ii) potentiometric titration; and (iii) ATR-FTIR spectroscopy. Potentiometric titration data analyzed using chemical speciation software are related to measured electrophoretic mobilities at the pH of interest. Titration of bacterial cells is used to identify the major proton-active functional groups and the overall concentration of these cell surface ligands at the cell membrane. Analysis of titration data shows notable differences between strains and conditions, confirming the appropriateness of this tool for an overall charge characterization. ATR-FTIR spectroscopy of whole cells is used to further characterize the bacterial biochemical composition and macromolecular structures that might be involved in the development of the net surficial charge of the organisms examined. The evaluation of the integrated intensities of HPO(2)(-) and carbohydrate absorption bands in the IR spectra reveals clear differences between growth protocols. Taken together, the three techniques seem to indicate that the dissolved oxygen tension during cell growth or acclimation can noticeably influence the expression of cell surface molecules and the measurable cell surface charge, though in a strain-dependent fashion.

Short-term inactivation rates of selected Gram-positive and Gram-negative bacteria attached to metal oxide mineral surfaces: role of solution and surface chemistry.

Metal oxides such as ferric or aluminum oxides can play an important role in the retention of bacteria in granular aquatic environments; however, their role in bacterial inactivation is not well understood. Herein, we examined the role of water chemistry and surface chemistry on the short-term inactivation rates of three bacteria when adhered to surfaces. To evaluate the role of water chemistry on the inactivation of attached bacteria, the loss in membrane integrity of bacteria attached to an iron oxide (Fe2O3) surface was measured over a range of water ionic strengths of either monovalent or divalent salts in the absence of a growth substrate. The influence of surface chemistry on the inactivation of attached bacteria was examined by measuring the loss in membrane integrity of cells attached to three surfaces (SiO2, Fe2O3, and Al2O3) at a specific water chemistry (10 mM KCl). Bacteria were allowed to attach onto the SiO2 or metal oxide coated slides mounted in a parallel-plate flow cell, and their inactivation rate (loss in membrane integrity) was measured directly without removing the cells from the surface and without disturbing the system. X-ray photoelectron spectroscopy analysis revealed a high correlation between the amounts of C-metal or O-metal bonds and the corresponding bacterial inactivation rates for each surface. Finally, for all three surfaces, a consistent increase in inactivation rate was observed with the type of bacterium in the order: Enterococcus faecalis, Escherichia coli O157:H7, and Escherichia coli D21f2.

Transport, motility, biofilm forming potential and survival of Bacillus subtilis exposed to cold temperature and freeze-thaw.

In cold climate regions, microorganisms in upper layers of soil are subject to low temperatures and repeated freeze-thaw (FT) conditions during the winter. We studied the effects of cold temperature and FT cycles on the viability and survival strategies (namely motility and biofilm formation) of the common soil bacterium and model pathogen Bacillus subtilis. We also examined the effect of FT on the transport behavior of B. subtilis at two solution ionic strengths (IS: 10 and 100 mM) in quartz sand packed columns. Finally, to study the mechanical properties of the bacteria-surface bond, a quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor changes in bond stiffness when B. subtilis attached to a quartz substrate (model sand surface) under different environmental conditions. We observed that increasing the number of FT cycles decreased bacterial viability and that B. subtilis survived for longer time periods in higher IS solution. FT treatment decreased bacterial swimming motility and the transcription of flagellin encoding genes. Although FT exposure had no significant effect on the bacterial growth rate, it substantially decreased B. subtilis biofilm formation and correspondingly decreased the transcription of matrix production genes in higher IS solution. As demonstrated with QCM-D, the bond stiffness between B. subtilis and the quartz surface decreased after FT. Moreover, column transport studies showed higher bacterial retention onto sand grains after exposure to FT. This investigation demonstrates how temperature variations around the freezing point in upper layers of soil can influence key bacterial properties and behavior, including survival and subsequent transport.

Inhibition of bacterial motility and spreading via release of cranberry derived materials from silicone substrates.

The motility of bacteria plays a key role in their colonization of surfaces during infection. Derivatives of cranberry fruit have been shown to interfere with bacterial motility. Herein, we report on the incorporation of cranberry derived materials (CDMs) into silicone substrates with the aim of impairing bacterial pathogen motility and spreading on the substrate surface. The release of CDMs from the silicone substrates when soaking in an aqueous medium was quantified for a period of 24h. Next, we showed that CDMs released from two silicone substrates remain bioactive as they downregulate the expression of the flagellin gene of two key uropathogens - Escherichia coli CFT073 and Proteus mirabilis HI4320. Furthermore, we demonstrate that CDM-modified silicone inhibits the swarming motility of P. mirabilis, an aggressive swarmer. The bioactive, CDM-modified substrates can find broad applications in the medical device and food industries where the impairment of bacterial colonization of surfaces is of paramount importance.

Effect of gold nanoparticles on extracellular nutrient-cycling enzyme activity and bacterial community in soil slurries: role of nanoparticle size and surface coating

Engineered nanoparticles (ENPs) are introduced to the soil environment mainly via wastewater biosolids applied to soils and their targeted delivery in agricultural applications. The impact of ENP size and surface coating on the activity of extracellular enzymes and bacterial community composition of an agricultural soil was examined using model gold nanoparticles (nAu). These endpoints were chosen as indicators of the soils response to external disturbances. The activity of five extracellular enzymes important in nutrient-cycling was measured in soils treated with commercially available 50 nm citrate-coated nAu and polyvinylpyrrolidone (PVP)-coated nAu of three different nominal diameters: 5, 50, and 100 nm. At low particle concentration (0.1 mg nAu kg−1 soil), decreasing the size of PVP-nAu resulted in an increased stimulation of soil enzyme activity. No correlation between size of PVP-nAu and soil enzyme activity was observed at a higher dose (100 mg nAu kg−1 soil). At a fixed size of 50 nm, citrate-coated nAu generally resulted in significant increases in soil enzyme activity at 30 days of exposure compared to PVP-coated nAu. Data from 16S rRNA gene sequencing showed that the community composition of soil spiked with citrate-coated nAu clustered significantly away from soil spiked with PVP-nAu at higher concentration (100 mg nAu kg−1 soil), showing the effect of type of nAu surface coatings. Abundance of bacteria annotated to operational taxonomic units (OTUs) from important soil bacterial groups, including Actinobacteria and Proteobacteria, increased following exposure of soil to nAu and more substantially following exposure to citrate-coated nAu for 30 days. This study shows that release of nanomaterials such as nAu to soils may have significant effects on soil enzyme activities and microbial communities and thus impact nutrient cycling in soils. Moreover, this study provides baseline knowledge that may be useful in customizing nanoparticle size and surface coating for their potential use as nutrient delivery agents in agriculture.

Amendment of Agricultural Soil with Metal Nanoparticles: Effects on Soil Enzyme Activity and Microbial Community Composition

Several types of engineered nanoparticles (ENPs) are being considered for direct application to soils to reduce the application and degradation of pesticides, provide micronutrients, control pathogens, and increase crop yields. This study examined the effects of different metal ENPs and their dissolved ions on the microbial community composition and enzyme activity of agricultural soil amended with biosolids. The activity of five extracellular nutrient-cycling enzymes was measured in biosolid-amended soils treated with different concentrations (1, 10, or 100 mg ENP/kg soil) of silver (nAg), zinc oxide (nZnO), copper oxide (nCuO), or titanium dioxide (nTiO2) nanoparticles and their ions over a 30-day period. At 30 days, nZnO and nCuO either had no significant effect on soil enzyme activity or enhanced enzyme activity. In contrast, Ag inhibited selected enzymes when dosed in particulate or dissolved form (at 100 mg/kg). nTiO2 either had no significant effect or slightly decreased enzyme activity. Illumina MiSeq sequencing of microbial communities indicated a shift in soil microbial community composition upon exposure to high doses of metal ions or nAg and negligible shift in the presence of nTiO2. Some taxa responded differently to nAg and Ag+. This work shows how metal ENPs can impact soil enzyme activity and microbial community composition upon introduction into soils amended with biosolids, depending on their type, concentration, and dissolution behavior, hence providing much needed information for the sustainable application of nanotechnology in agriculture.