Search

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.

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.

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.

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.

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., (Copyright 1999 Academic Press.)

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.

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.

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.