Fosfomycin enhances the active transport of tobramycin in Pseudomonas aeruginosa.

Elevated levels of mucins present in bronchiectatic airways predispose patients to bacterial infections and reduce the effectiveness of antibiotic therapies by directly inactivating antibiotics. Consequently, new antibiotics that are not inhibited by mucins are needed to treat chronic respiratory infections caused by Pseudomonas aeruginosa and Staphylococcus aureus. In these studies, we demonstrate that fosfomycin synergistically enhances the activity of tobramycin in the presence of mucin. The bactericidal killing of a novel 4:1 (wt/wt) combination of fosfomycin-tobramycin (FTI) is superior (>9 log(10) CFU/ml) relative to its individual components fosfomycin and tobramycin. Additionally, FTI has a mutation frequency resulting in an antibiotic resistance >3 log(10) lower than for fosfomycin and 4 log(10) lower than for tobramycin for P. aeruginosa. Mechanistic studies revealed that chemical adducts are not formed, suggesting that the beneficial effects of the combination are not due to molecular modification of the components. FTI displayed time-kill kinetics similar to tobramycin and killed in a concentration-dependent fashion. The bactericidal effect resulted from inhibition of protein biosynthesis rather than cell wall biosynthesis. Studies using radiolabeled antibiotics demonstrated that tobramycin uptake was energy dependent and that fosfomycin enhanced the uptake of tobramycin in P. aeruginosa in a dose-dependent manner. Lastly, mutants resistant to fosfomycin and tobramycin were auxotrophic for specific carbohydrates and amino acids, suggesting that the resistance arises from mutations in specific active transport mechanisms. Overall, these data demonstrate that fosfomycin enhances the uptake of tobramycin, resulting in increased inhibition of protein synthesis and ultimately bacterial killing.

Extraction of MS2 phage RNA from upper respiratory tract specimens by use of flat glass devices.

The isolation of pure nucleic acids from clinical samples is a crucial step in the molecular diagnosis of viral infections by nucleic acid testing (NAT). In this study, novel flat glass devices (cards) were demonstrated to support the rapid and efficient extraction of nucleic acids from upper respiratory tract specimens (nasal washes and swabs). The performance of the nucleic acid extraction cards was directly compared to an existing standardized and automated platform for viral extraction from these types of specimens. The flowthrough card method improved the speed of nucleic acid purification and accommodated larger sample volumes in extraction of bacteriophage MS2 RNA from the various specimen matrices. The dynamic range and estimated sensitivity of the card extraction method for reverse transcriptase quantitative real-time PCR (RT-qPCR)-based detection approximate those of the standardized magnetic glass bead extraction method used in this study.

Multiple pH measurement during storage may detect bacterially contaminated platelet concentrates.

BACKGROUND: Bacterial contamination or platelet (PLT) metabolism can change the pH of stored PLT concentrates (PCs). Measurement of pH for quality control is currently done on a limited basis. An easy noninvasive method was developed to obtain sequential pH measurements over time, without risking contamination and/or consuming PCs. STUDY DESIGN AND METHODS: The objective was to measure pH profiles of bacterially contaminated PCs over 7 days of storage. Small-volume PC storage bags with incorporated pH sensor were prepared and in vitro variables were tested using aliquots of PCs. The pH sensors were used to delineate trends associated with the deterioration of these PCs upon inoculation with 19 different bacterial strains and one yeast. RESULTS: Monitoring the pH trends in real time in a noninvasive fashion, most bacterial strains were detected within 24 to 72 hours after spiking into the bag. At the time of detection, bacterial concentrations had reached levels between 1×10(3) and 1×10(8) colony-forming units/mL. Several strains had pH rebound after initial drop. Multiple noninvasive pH reads allowed bacterial detection whereas single pH reads could give false-negative results. CONCLUSIONS: The noninvasive pH sensor facilitated the detection of most strains of bacterial contaminants within 3 days with no potential for sampling error.

Antibacterial activities of a fosfomycin/tobramycin combination: a novel inhaled antibiotic for bronchiectasis.

OBJECTIVES: To compare the in vitro and in vivo activities of a 4:1 (w/w) fosfomycin/tobramycin combination (FTI) with those of fosfomycin and tobramycin alone against cystic fibrosis (CF) and non-CF bronchiectasis pathogens. METHODS: Clinical isolates of CF Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Stenotrophomonas maltophilia, Burkholderia cepacia complex, Escherichia coli and Klebsiellia spp. were evaluated by MIC, MBC, post-antibiotic effect (PAE), synergy, time-kill, a rat pneumonia model and spontaneous mutation frequency (SMF). RESULTS: FTI showed high activity against E. coli, H. influenzae, S. aureus and Klebsiella spp. For the S. aureus strains, 75% of which were methicillin resistant (MRSA), FTI had a lower MIC(90) than tobramycin. For P. aeruginosa, FTI had a lower MIC(90) than fosfomycin, but tobramycin was more active than either. Synergy studies showed no antagonism between fosfomycin and tobramycin, and 93% of the isolates demonstrated no interaction. FTI was rapidly bactericidal and exhibited concentration-dependent killing in time-kill studies. In the rat pneumonia model, FTI and tobramycin demonstrated bactericidal killing of P. aeruginosa; both were more active than fosfomycin alone. The SMF for S. aureus resistance to FTI was 2-4 log(10) lower than that for tobramycin and 2-7 log(10) lower than that for fosfomycin. For P. aeruginosa, the FTI SMF was 2-3 log(10) lower than that for fosfomycin and 1-2 log(10) lower than that for tobramycin. CONCLUSIONS: FTI is a broad-spectrum antibiotic combination with high activity in vitro and in vivo. These data suggest FTI could be a potential treatment for respiratory infections caused by gram-positive and gram-negative aerobic bacteria.

Noninvasive measurement of pH in platelet concentrates with a fiber optic fluorescence detector.

BACKGROUND: Stored platelets (PLTs) are metabolically active, resulting in a decrease of pH during storage. The pH of PLT concentrates (PCs) is recognized as a measure of quality, and pH limits are set by regulatory bodies. A pH sensor was built into a PLT storage container, and the feasibility of testing pH using a noninvasive fluorescent measurement method was evaluated. STUDY DESIGN AND METHODS: A citrated polyvinylchloride (PVC) PLT storage container with pH sensor insert was made and evaluated for biocompatibility during PLT storage and on pH reading accuracy, reproducibility, and durability. A noninvasive fluorescence reader was tested versus syringe-based sampling and subsequent measurement with a blood gas analyzer (BGA). The effect of interfering substances in plasma on the accuracy of this optical measurement was tested. Calibration and accuracy of the pH sensor were determined in both phosphate-buffered saline and in PCs. RESULTS: The citrated PVC storage container with pH sensor insert showed good storage properties for 300 mL of pooled buffy coat PLTs in plasma over 7 days. The pH sensor was easy to use and tracked pH(22) in the range of 6.2 to 7.8 over 11 days of storage. Accuracy in PCs was 0.08 pH units measured at 22 degrees C when calibrated against a BGA. CONCLUSION: The storage container with integrated pH sensor and noninvasive reader allows pH of PCs to be tracked over time in a noninvasive manner.