Sub-Picogram, Inline Detection Of Proteins Using Microfluidic Drop Generators And Shape-Based Detection

Proteomic workflows rely on sensitive and precise methods for purifying and detecting proteins, and one of the most popular methods is liquid chromatography (LC) followed by an inline detector. However, the most commonly used method, UV-Vis absorbance, provides relatively low sensitivity and requires long path lengths in the flow cell. In this thesis, we present a highly sensitive, label-free method to detect proteins in continuous flow, using a pressure-driven microfluidic droplet generator. The system consists of a cross junction or flow focusing structure where the flowing stream of proteins is combined with two coflowing streams of oil which break up the former stream into a train of droplets. In such a system, the droplet size predictably scales with interfacial tension (IFT) of the oil-water interface. When globular proteins (eg BSA) enter the junction, they adsorb to the interface and reduce IFT, and thereby the droplet radius. Temporal variations in droplet size therefore correlate with changes in protein concentration. Using microfluidic drop generators as an inline detection method provides two benefits: 1) it can improve available limits of detection, because the high surface area to volume ratio enhances adsorption phenomena, and 2) it provides fast time resolution, due to the high rate of drop generation. Four different carrier phases (oleic acid, octanol, hexadecane, and FC-40) were tested with four proteins (BSA, thyroglobulin, uridine, placental growth factor, galectin and blood plasma). Droplet sizes were found to decrease when a hydrophobic protein was combined with a hydrophobic carrier. The best limit of detection is ~1 μg/mL in a 1 nL droplet, which equates to 1 fg of total protein. The low detection limits are due to favorable scaling: the high surface area to volume ratio in droplet systems increases the probability of adsorption, and therefore changes drop size even at low concentration. When used as a detector for high performance liquid chromatography (HPLC), it demonstrates sensitivity up to 100X better than conventional UV-Vis detectors. This detection method can potentially serve as a label-free, universal detector for proteins

Activity of the Lactate Dehydrogenase Inhibitor Oxamic Acid against the Fermentative Bacterium \u3ci\u3eStreptococcus mitis/oralis\u3c/i\u3e: Bactericidal Effects and Prevention of Daptomycin Resistance In Vitro and in an Ex Vivo Model

Streptococcus mitis/oralis is a fermentative bacterium that relies on lactate dehydrogenase to balance its redox poise and keep glycolysis active. Metabolomic analysis of an in vitro– derived daptomycin-resistant (DAP-R) S. mitis/oralis strain (351-D10) revealed differences in glucose catabolism relative to its DAP-susceptible (DAP-S) parental strain, 351. Metabolic changes associated with the transition to this DAP-R phenotype suggested that inhibiting glycolysis could alter DAP susceptibility. In addition, the strong reliance of S. mitis/oralis on glycolysis for energy and biosynthetic intermediates suggested that inhibiting glycolysis would adversely affect growth and biomass accumulation. To test these hypotheses, we used the lactate dehydrogenase inhibitor oxamic acid (OXA) to assess its efficacy against DAP-S S. mitis/oralis strain 351 during DAP exposures in vitro and ex vivo. As expected, OXA was growth inhibitory to S. mitis/oralis in a dose-dependent manner in vitro; however, it did not alter in vitro DAP susceptibility profiles. In contrast, OXA did prevent the emergence of DAP-R in an ex vivo model of simulated endocardial vegetations. These data suggest that metabolic inhibitors directed against this fermentative bacterium with limited metabolic capabilities could enhance killing and potentially forestall the emergence of DAP resistance

Targeting Dalbavancin Inoculum Effect: Adjunctive Single Dose of Daptomycin.

INTRODUCTION: Daptomycin (DAP) has proven to be a viable alternative amid vancomycin resistance; however, the use of DAP post vancomycin treatment has led to the development of DAP non-susceptible (DNS) strains. Dalbavancin (DAL), a novel single-dosed lipoglycopeptide, has shown enhanced activity against highly resistant Staphylococcus aureus strains. However, on the basis of previous reports and our observations, DAL does not demonstrate similar activity at high versus low inoculum levels. Therefore, we hypothesized that addition of DAP even at minimal concentrations (single dose on day 1) will lower the inoculum to the level that can be cleared by dalbavancin. METHODS: Isolates from methicillin-resistant S. aureus (MRSA)-infected patients with varying susceptibility profiles were evaluated using broth microdilution methods. Two DNS-VISA strains (vancomycin intermediate resistant S. aureus) and one MRSA strain were further evaluated in a one-compartment PK/PD model using a high starting initial inoculum of 109 CFU/mL as well as low initial inoculum of 107 CFU/mL over 168 h to assess the activity of DAL and DAP monotherapy and in combination. RESULTS: Single therapies were not bactericidal when evaluated in the 168 h in vitro one-compartment model with an initial inoculum of 109; however, the combination of DAL plus single dose of DAP resulted in enhanced killing at the end of the 168-h exposure. DAL single therapy caused reduction in colony counts down to detection limit (2 log10 CFU/ml) at a lower inoculum but did not show enhancement (< 2 log10 CFU/ml) at higher initial inoculums (P < 0.01) for all three strains. Similarly, DAP caused initial bacterial reduction up to 4 log10 CFU/ml with regrowth at about 32 h of exposure, which stayed at initial inoculum levels for the duration of the model for all three strains. CONCLUSIONS: Dalbavancin inoculum effect is a major issue in bacterial infections with high bacterial loads and the combination of DAL plus single dose of DAP showed promise in eradicating resistant S. aureus strains at high inoculums

Translating phage therapy into the clinic: Recent accomplishments but continuing challenges.

Phage therapy is a medical form of biological control of bacterial infections, one that uses naturally occurring viruses, called bacteriophages or phages, as antibacterial agents. Pioneered over 100 years ago, phage therapy nonetheless is currently experiencing a resurgence in interest, with growing numbers of clinical case studies being published. This renewed enthusiasm is due in large part to phage therapy holding promise for providing safe and effective cures for bacterial infections that traditional antibiotics acting alone have been unable to clear. This Essay introduces basic phage biology, provides an outline of the long history of phage therapy, highlights some advantages of using phages as antibacterial agents, and provides an overview of recent phage therapy clinical successes. Although phage therapy has clear clinical potential, it faces biological, regulatory, and economic challenges to its further implementation and more mainstream acceptance

Biofilm Time-Kill Curves to Assess the Bactericidal Activity of Daptomycin Combinations against Biofilm-Producing Vancomycin-Resistant Enterococcus faecium and faecalis

Introduction: E. faecium and E. faecalis are responsible for 13.9% of hospital-acquired infections with frequent resistance to vancomycin (82.6% of E. faecium, 9.5% of E. faecalis). Medical device infections secondary to enterococci often require combination therapy due to impaired activity against biofilm embedded cells. In vitro data demonstrate synergistic activity of daptomycin combinations. Using a novel, biofilm time-kill approach, we evaluated whether daptomycin combinations maintained synergy against biofilm-producing E. faecium and E. faecalis. Methods: Broth microdilution (BMD) and biofilm MIC (bMIC) values for daptomycin, ampicillin, ceftriaxone, fosfomycin, and rifampin were determined against biofilm-producing E. faecium and E. faecalis. Daptomycin combination bMIC values were determined in the presence of biologic concentrations of other antimicrobials. Synergy was evaluated against two E. faecalis (R6981, R7808) and two E. faecium (5938 and 8019) using a previously described biofilm time-kill method. Synergy was defined as ≥2 log10 CFU/cm2 reduction over the most active agent alone. Bactericidal activity was defined as ≥3 log10 CFU/cm2 reduction. Results: Daptomycin bMICs were 2–8-fold higher than BMD. In the presence of other antimicrobials, daptomycin bMICs were reduced ≥ two-fold in dilutions. Ceftriaxone and ampicillin demonstrated the most potent combinations with daptomycin, yielding synergy against three of four strains. Daptomycin plus rifampin was synergistic against E. faecium 5938 and E. faecalis 6981 and produced bactericidal kill. The combination of daptomycin plus fosfomycin displayed synergy solely against E. faecalis 6981. Conclusions: Daptomycin combinations with beta-lactams demonstrated promising synergistic activity against both E. faecium and E. faecalis. While daptomycin plus rifampin yielded bactericidal results, the effect was not seen across all organisms. These combinations warrant further evaluation to determine the optimal dose and response

Activity of the Lactate Dehydrogenase Inhibitor Oxamic Acid against the Fermentative Bacterium Streptococcus mitis/oralis: Bactericidal Effects and Prevention of Daptomycin Resistance In Vitro and in an Ex Vivo Model

Streptococcus mitis/oralis is a fermentative bacterium that relies on lactate dehydrogenase to balance its redox poise and keep glycolysis active. Metabolomic analysis of an in vitro–derived daptomycin-resistant (DAP-R) S. mitis/oralis strain (351-D10) revealed differences in glucose catabolism relative to its DAP-susceptible (DAP-S) parental strain, 351. Metabolic changes associated with the transition to this DAP-R phenotype suggested that inhibiting glycolysis could alter DAP susceptibility. In addition, the strong reliance of S. mitis/oralis on glycolysis for energy and biosynthetic intermediates suggested that inhibiting glycolysis would adversely affect growth and biomass accumulation. To test these hypotheses, we used the lactate dehydrogenase inhibitor oxamic acid (OXA) to assess its efficacy against DAP-S S. mitis/oralis strain 351 during DAP exposures in vitro and ex vivo. As expected, OXA was growth inhibitory to S. mitis/oralis in a dose-dependent manner in vitro; however, it did not alter in vitro DAP susceptibility profiles. In contrast, OXA did prevent the emergence of DAP-R in an ex vivo model of simulated endocardial vegetations. These data suggest that metabolic inhibitors directed against this fermentative bacterium with limited metabolic capabilities could enhance killing and potentially forestall the emergence of DAP resistance

Evaluation of Omadacycline Alone and in Combination with Rifampin against Staphylococcus aureus and Staphylococcus epidermidis in an In Vitro Pharmacokinetic/Pharmacodynamic Biofilm Model.

Biofilm-associated infections lead to substantial morbidity. Omadacycline (OMC) is a novel aminomethylcycline with potent in vitro activity against Staphylococcus aureus and Staphylococcus epidermidis, but data surrounding its use in biofilm-associated infections are lacking. We investigated the activity of OMC alone and in combination with rifampin (RIF) against 20 clinical strains of staphylococci in multiple in vitro biofilm analyses, including an in vitro pharmacokinetic/pharmacodynamic (PK/PD) CDC biofilm reactor (CBR) model (simulating human exposures). The observed MICs for OMC demonstrated potent activity against the evaluated strains (0.125 to 1 mg/L), with an increase of MICs generally observed in the presence of biofilm (0.25 to >64 mg/L). Furthermore, RIF was shown to reduce OMC biofilm MICs (bMICs) in 90% of strains, and OMC plus RIF combination in biofilm time-kill analyses (TKAs) exhibited synergistic activity in most of the strains. Within the PK/PD CBR model, OMC monotherapy primarily displayed bacteriostatic activity, while RIF monotherapy generally exhibited initial bacterial eradication, followed by rapid regrowth likely due to the emergence of RIF resistance (RIF bMIC, >64 mg/L). However, the combination of OMC plus RIF produced rapid and sustained bactericidal activity in nearly all the strains (3.76 to 4.03 log10 CFU/cm2 reductions from starting inoculum in strains in which bactericidal activity was reached). Furthermore, OMC was shown to prevent the emergence of RIF resistance. Our data provide preliminary evidence that OMC in combination with RIF could be a viable option for biofilm-associated infections with S. aureus and S. epidermidis. Further research involving OMC in biofilm-associated infections is warranted