3× multiplexed detection of antibiotic resistant plasmids with single molecule sensitivity.

Bacterial pathogens resistant to antibiotics have become a serious health threat. Those species which have developed resistance against multiple drugs such as the carbapenems, are more lethal as these are last line therapy antibiotics. Current diagnostic tests for these resistance traits are based on singleplex target amplification techniques which can be time consuming and prone to errors. Here, we demonstrate a chip based optofluidic system with single molecule sensitivity for amplification-free, multiplexed detection of plasmids with genes corresponding to antibiotic resistance, within one hour. Rotating disks and microfluidic chips with functionalized polymer monoliths provided the upstream sample preparation steps to selectively extract these plasmids from blood spiked with E. coli DH5α cells. Waveguide-based spatial multiplexing using a multi-mode interference waveguide on an optofluidic chip was used for parallel detection of three different carbapenem resistance genes. These results point the way towards rapid, amplification-free, multiplex analysis of antibiotic-resistant pathogens.

Formation of eLiposomes as a drug delivery vehicle.

This paper discusses the formation of eLiposomes, defined as a liposome with internal emulsion droplets. Liposomes have been investigated as passively targeted drug carriers due to their ability to deliver drugs to a cancerous tumor via the enhanced permeability and retention (EPR) effect. The enclosed emulsion droplets in an eLiposome add the ability to further control the location and time of release from the liposome with ultrasound. Emulsion droplets were formed from perfluorohexane (PFC6) by sonication at 20 kHz and stabilized with dipalmitoyl phosphatidyl choline (DPPC). The size of the resulting droplets was reduced to approximately 100 nm or 50 nm by extrusion through polycarbonate filters of the same size at 50°C. Small unilamellar vesicles (SUVs) were prepared from DPPC by thin film hydration and extrusion through a 50 nm filter. Interdigitated DPPC sheets were prepared from the SUVs by the addition of ethanol to a concentration of 3M. Excess ethanol was removed by centrifugation washing. The sheets were mixed with emulsion and the solution was heated to 50°C, resulting in the refolding of the DPPC sheets into closed vesicles. Emulsion droplets were encapsulated inside of the newly formed eLiposomes. The size of the eLiposomes was reduced by extrusion. Cryogenic transmission electron microscopy (cryoTEM) and negative-staining TEM were used to image the emulsion droplets and the eLiposomes. Encapsulation of emulsion droplets was verified by rotating the microscope stage of cryoTEM samples.

Swellable coatings for hearing aid applications.

The problem of acoustic feedback in hearing aids could be solved potentially by applying a compliant hydrogel to the outer surface that would conform to the ear canal and block feedback. With this objective, several formulations of hydrogels were developed and their swelling and mechanical properties investigated. Hydrogel formulations were polymerized from hydroxyethyl methacrylate (HEMA) and N-vinyl-pyrrolidone (NVP), with various photo-initiators, crosslinkers, and swelling agents. The hydrogel that swelled most rapidly and yet remained undissolved in water had a monomer composition of 40 mol% HEMA, 60 mol% NVP, with 1 wt% polyethylene glycol dimethacrylate as a crosslinker, and 0.5 wt% 2,2-dimethoxy-2-phenyl-acetophenone as the photo-initiator. The tensile modulus, strength, hardness, and durability of the dry hydrogels were not a strong function of composition. In the swollen state, the mechanical properties were much reduced. The potential use of these materials on hearing aids has been discussed in this article.

Effect of pulsed ultrasound in combination with gentamicin on bacterial viability in biofilms on bone cements in vivo.

AIMS: The aim of this study is to investigate whether pulsed ultrasound (US) in combination with gentamicin yields a decreased viability of bacteria in biofilms on bone cements in vivo. METHODS AND RESULTS: Bacterial survival on bone cement in the presence and absence of ultrasound was compared in a rabbit model. Two bone cement samples with an Escherichia coli ATCC 10798 biofilm were implanted in a total of nine rabbits. In two groups bone cement discs loaded with gentamicin, freshly prepared and aged were used, and in one group unloaded bone cement discs in combination with systemically administered gentamicin. Pulsed ultrasound with a frequency of 28.48 kHz and a maximum acoustic intensity of 500 mW cm(-2) was applied continuously from 24 h till 72 h postsurgery on one of the two implanted discs. After euthanization and removal of the bacteria from the discs, the number of viable bacteria were quantified and skin samples were analysed for histopathological examination. Application of ultrasound, combined with gentamicin, reduced the viability of the biofilms in all three groups varying between 58 and 69% compared with the negative control. Histopathological examinations showed no skin lesions. CONCLUSIONS: Ultrasound resulted in a tendency of improved efficacy of gentamicin, either applied locally or systemically. Usage of ultrasound in this model proved to be safe. SIGNIFICANCE AND IMPACT OF THE STUDY: This study implies that ultrasound could improve the prevention of infection immediately after surgery, especially because the biomaterials, gentamicin and ultrasound used in this model are all in clinical usage, but not yet combined in clinical practice.

Ultrasonically enhanced vancomycin activity against Staphylococcus epidermidis biofilms in vivo.

Infection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.

Factors affecting acoustically triggered release of drugs from polymeric micelles.

A custom ultrasonic exposure chamber with real-time fluorescence detection was used to measure acoustically-triggered drug release from Pluronic P-105 micelles under continuous wave (CW) or pulsed ultrasound in the frequency range of 20 to 90 kHz. The measurements were based on the decrease in fluorescence intensity when drug was transferred from the micelle core to the aqueous environment. Two fluorescent drugs were used: doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb). Pluronic P-105 at various concentrations in aqueous solutions was used as a micelle-forming polymer. Drug release was most efficient at 20-kHz ultrasound and dropped with increasing ultrasonic frequency despite much higher power densities. These data suggest an important role of transient cavitation in drug release. The release of DOX was higher than that of Rb due to stronger interaction and deeper insertion of Rb into the core of the micelles. Drug release was higher at lower Pluronic concentrations, which presumably resulted from higher local drug concentrations in the core of Pluronic micelles when the number of micelles was low. At constant frequency, drug release increased with increasing power density. At constant power density and for pulse duration longer than 0.1 s, peak release under pulsed ultrasound was the same as stationary release under CW ultrasound. Released drug was quickly re-encapsulated between the pulses of ultrasound, which suggests that upon leaving the sonicated volume, the non-extravasated and non-internalized drug would circulate in the encapsulated form, thus preventing unwanted drug interactions with normal tissues.

DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study.

To minimize adverse side effects of chemotherapy, we have developed a micellar drug carrier that retains hydrophobic drugs, and then releases the drug by ultrasonic stimulation. This study investigated the DNA damage induced by doxorubicin (DOX) delivered to human leukemia (HL-60) cells from pluronic P-105 micelles with and without the application of ultrasound. The comet assay was used to quantify the amount of DNA damage. No significant DNA damage was observed when the cells were treated with 0.1, 1 and 10 wt% P-105 with or without ultrasound (70 kHz, 1.3 W/cm(2)) for 1 h or for up to 3 h in 10 wt% P-105. However, when cells were incubated with 10 microg/ml free DOX for up to 9 h, DNA damage increased with incubation time (P=0.0011). Exposure of cells to the same concentration of DOX in the presence of 10-wt% P-105 showed no significant DNA damage for up to 9 h of incubation. However, when ultrasound was applied, a rapid and significant increase in DNA damage was observed (P=0.0001). The application of ultrasound causes the release of DOX from micelles or causes the HL-60 cells to take up the micelle encapsulated DOX. Our experiments indicated that the combination of DOX, ultrasound and pluronic P105 causes the largest DNA damage to HL-60 cells. We believe that this technique can be used for controlled drug delivery.

Pulsed ultrasound enhances the killing of Escherichia coli biofilms by aminoglycoside antibiotics in vivo.

Escherichia coli biofilms on two polyethylene disks were implanted subcutaneously into rabbits receiving systemic gentamicin. Ultrasound was applied for 24 h to one disk. Both disks were removed, and viable bacteria were counted. Pulsed ultrasound significantly reduced bacterial viability below that of nontreated biofilms without damage to the skin.

Attachment of hyaluronic acid to polypropylene, polystyrene, and polytetrafluoroethylene.

Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH3 to aminate the polymer surface and were subsequently reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.

Investigation of the mechanism of the bioacoustic effect.

Bacterial biofilms growing on implanted medical devices are difficult to eradicate, even with aggressive antibiotic therapy. However, application of ultrasound enhances the effectiveness of the antibiotic. The possible mechanisms of this phenomenon were explored in light of the observed influence of various ultrasonic parameters on the enhanced action of gentamicin against biofilms of Pseudomonas aeruginosa. It is postulated that ultrasound increases the transport of gentamicin through the cell membranes, which is the proposed rate determining step in killing by gentamicin. It is possible that the ultrasound perturbs the cell membrane and stimulates active uptake or permits passive uptake by temporarily disrupting the membrane or other structural cell components. The cell membrane disruption could be caused by high pressure, high shear stress, or cavitation. The dependence upon peak power density suggests that acoustic pressure plays a significant role. There is also a strong frequency component that causes the killing effect to decrease as frequency increases. A mathematical analysis of oscillatory shear stress on the cell shows that the magnitude of stress increases with frequency; thus, the hypothesis of oscillatory shear inducing antibiotic uptake is discounted. In addition, the shear displacement caused by shear forces is very small, so the shear disruption caused by oscillatory flow in an acoustic field has minimal impact. The experimental data also rule out the existence of transient cavitation in the bioacoustic effect. It is possible that stable cavitation and the accompanying microstreaming contribute to the bioacoustic effect.

Ultrasonic enhancement of antibiotic action on Escherichia coli biofilms: an in vivo model.

Biofilm infections are a common complication of prosthetic devices in humans. Previous in vitro research has determined that low-frequency ultrasound combined with aminoglycoside antibiotics is an effective method of killing biofilms. We report the development of an in vivo model to determine if ultrasound enhances antibiotic action. Two 24-h-old Escherichia coli (ATCC 10798) biofilms grown on polyethylene disks were implanted subcutaneously on the backs of New Zealand White female rabbits, one on each side of the spine. Low-frequency (28.48-kHz) and low-power-density (100- and 300-mW/cm2) continuous ultrasound treatment was applied for 24 h with and without systemic administration of gentamicin. The disks were then removed, and the number of viable bacteria on each disk was determined. At the low ultrasonic power used in this study, exposure to ultrasound only (no gentamicin) caused no significant difference in bacterial viability. In the presence of antibiotic, there was a significant reduction due to 300-mW/cm2 ultrasound (P = 0.0485) but no significant reduction due to 100-mW/cm2 ultrasound. Tissue damage to the skin was noted at the 300-mW/cm2 treatment level. Further development of this technique has promise in treatment of clinical implant infections.

Micellar delivery of doxorubicin and its paramagnetic analog, ruboxyl, to HL-60 cells: effect of micelle structure and ultrasound on the intracellular drug uptake.

The effect of Pluronic P-105 micelle structure and ultrasound on the uptake of two anthracycline drugs, doxorubicin and its paramagnetic analogue, ruboxyl, by HL-60 cells was investigated. Pluronic micellization was studied over the temperature range of 25-42 degrees C using the EPR and fluorescence spectroscopy. In the presence of Pluronic P-105 at concentrations corresponding to unimers (or loose aggregates), drug uptake by HL-60 cells was enhanced, apparently due to the effect of the polymeric surfactant on cell membrane permeability. At Pluronic concentrations corresponding to the formation of dense micelles with hydrophobic cores, drug uptake was substantially decreased. However, insonation with 70 kHz ultrasound enhanced the intracellular uptake of drugs encapsulated in dense Pluronic micelles. These findings may provide for developing a new technique of drug targeting by encapsulating the drug in micelles to prevent unwanted interactions with healthy cells and focusing ultrasound on a tumor to enhance drug uptake at the tumor site.

Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound.

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls.

Reducing bacterial resistance to antibiotics with ultrasound.

The effect of erythromycin on planktonic cultures of Psedomonas aeruginosa, with and without application of 70 kHz ultrasound, was studied. Ultrasound was applied at levels that had no inhibitory effect on cultures of Ps. aeruginosa. Ultrasound in combination with erythromycin reduced the viability of Ps. aeruginosa by 1-2 orders of magnitude compared with antibiotic alone, even at concentrations below the minimum inhibitory concentration (MIC). Electron-spin resonance studies suggest that ultrasound induces uptake of antibiotic by perturbing or stressing the membrane. This application of ultrasound may be useful for expanding the number of drugs available for treating localized infections by rendering bacteria susceptible to normally ineffective antibiotics.

Treatment of bacterial biofilms on polymeric biomaterials using antibiotics and ultrasound.

New studies on the effect of frequency and duration of exposure upon the ultrasonic enhanced action of gentamicin against biofilms of Escherichia coli are presented in this paper. Ultrasound was applied at clinical levels which would not harm human tissue. The study on frequency indicated that lower frequencies produce higher levels of killing. The results of the timed experiment indicate that complete sterilization of a 14-h biofilm can be achieved after 6 h of exposure. These findings are significant because they show that biofilms can be reduced to zero reproductive ability as assessed by plate counting.

Ultrasonic activated drug delivery from Pluronic P-105 micelles.

Pluronic copolymer P-105 at micellar concentration of 10 wt% was found to increase the activity of the anti-cancer drug, doxorubicin (DOX) against HL-60 cells. Despite the enhanced activity, drug uptake by the cells from P-105 micelles (measured by fluorescence) was found to be much lower than that from a molecular solution of DOX (without the surfactant). Ultrasound (US) was applied as a tool to release drug from the micelles. Based on the combination of ultrasound and micellar treatment, doxorubicin exhibited IC50 concentrations of 2.35, 0.9, 1.25, 0.19 microg/ml with respect to DOX, DOX/US, micellar DOX, and micellar DOX/US, respectively. The results suggest that by encapsulating the anti-cancer drug in micellar carriers and focussing ultrasound on the tumor site, a new approach to drug targetting can be developed.

Factors affecting the permeability of Pseudomonas aeruginosa cell walls toward lipophilic compounds: effects of ultrasound and cell age.

The objective of this research was to elucidate the factors effecting the permeability of cell membranes of gram-negative bacteria toward hydrophobic compounds. Ultrasound treatment, cell age, and the phase state of phospholipid membranes were considered. Spin-labeling EPR method was used to quantify the penetration and distribution of a lipophilic spin probe, 16-doxylstearic acid (16-DS), in Pseudomonas aeruginosa cell membranes. This bacterium was chosen because of its reported resistance to the action of hydrophobic antibiotics caused by the low permeability of the outer cell membrane for hydrophobic compounds. EPR spectra were collected from cell pellets and cell lysates. The overall spin probe uptake was measured in 10% SDS-cell lysates. Lysis with 0.6% SDS revealed the fraction of the probe located in membrane sites readily accessible to the surfactant. The results indicated a structural heterogeneity of P. aeruginosa membranes, with the presence of structurally "stronger" and "weaker" sites characterized by different susceptibility to the SDS treatment. The intracellular concentration of 16-DS was higher in insonated cells and increased linearly with the sonication power. EPR spectra indicated that ultrasound enhanced the penetration of the probe into the structurally stronger sites of the inner and outer cell membranes. The effect of ultrasound on the cell membranes was transient in that the initial membrane permeability was restored upon termination of the ultrasound treatment. These results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes. This resistance may be reduced by the simultaneous application of antibiotic and ultrasound. This hypothesis was confirmed in our experiments with P. aeruginosa exposed to erythromycin.

In vitro response of Escherichia coli to antibiotics and ultrasound at various insonation intensities.

Microbial infections on implanted medical devices are difficult to treat. Application of 70 kHz ultrasonic irradiation to gentamicin treatment greatly enhanced the action of the antibiotic in terms of reduced viable bacterial concentrations. Ultrasonic irradiation was carried out at various insonation intensities that were noninhibitory in the absence of antibiotic. Synergistic killing was observed to be a function of ultrasonic intensity. Greatest killing (approximately 5 log reduction in viable population) was realized at full intensity (4.5 W/cm2), and decreased with reductions in power density. At lowest intensity (10 mW/cm2), no significant acoustic enhanced killing was noted.

The effect of ultrasonic frequency upon enhanced killing of P. aeruginosa biofilms.

It is widely recognized that the bacteria sequestered in a biofilm on a medical implant are much more resistant to antibiotics than their planktonic counterparts. Recent studies have shown that application of antibiotic along with low power ultrasound significantly increases the killing of planktonic bacteria by the antibiotic. Herein is reported a similar application of antibiotic and ultrasound to sessile bacteria in biofilms of Pseudomonas aeruginosa on a polyethylene substrate. Biofilm viability was measured after exposure to 12 micrograms/ml gentamicin sulfate and 10 mW/cm2 ultrasound at frequencies of 70 kHz, 500 kHz, 2.25 MHz, and 10 MHz. The results indicate that a significantly greater fraction of the bacteria was killed by gentamicin when they were subjected to ultrasound. However, ultrasound by itself did not have any deleterious effect on the biofilm viability. In addition, lower-frequency insonation is significantly more effective than higher frequency in reducing bacterial viability within the biofilm. The possible mechanisms of synergistic action are discussed.

Measurement of bacterial growth rates on polymers.

A video microscope system and a mathematical model were developed to observe and model the early stage of bacterial growth on polymer surfaces. Glass slides were coated with polyorthoester, poly(L-lactic acid), and polysulfone, and inserted into a laminar flow cell to expose them to bacterial cultures of Staphylococcus epidermidis, Pseudomonas aeruginosa, or Escherichia coli. The free energy of adhesion (delta Fadh) was determined from contact-angle measurements. The microscopic observations along with the mathematical model allowed measurement of the rates of adhesion, release, and growth. The growth rate of P. aeruginosa on the various surfaces correlated to the delta Fadh. The growth rates of all species on all of the surfaces were slower than the growth rates of the bacteria in suspension. The mathematical model is valid for early growth before the bacteria form a complete monolayer, and is useful in predicting and modeling early growth of bacteria on implanted biomaterials.