In Vitro Antibiofilm Activity of Fosfomycin Alone and in Combination with Other Antibiotics against Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa.

BACKGROUND: Due to its rapid resistance development and ability to form biofilms, treatment of Pseudomonas aeruginosa infections is becoming more complicated by the day. Drug combinations may help reduce both resistance and biofilm formation. METHODS: Using the microtiter plate assay, we investigated the in vitro inhibition of biofilm formation and the disruption of preformed biofilms in multidrug-resistant and extensively drug-resistant clinical isolates of P. aeruginosa in the presence of peak plasma levels of eight antipseudomonal antibiotics alone and in combination with fosfomycin: ceftazidime, piperacillin/tazobactam, cefepime, imipenem, gentamicin, amikacin, ciprofloxacin and colistin. RESULTS: Combination therapy was significantly superior to monotherapy in its inhibition of biofilm formation. The highest inhibition rates were observed for combinations with colistin, cefepime and ceftazidime. CONCLUSION: Our results support fosfomycin combination therapy as an enhanced prophylactic option. Moreover, combinations with ß-lactam antibiotics and colistin demonstrated a more potent inhibition effect on biofilm formation than protein synthesis inhibitors.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies.

BACKGROUND: Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. METHODS: After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. RESULTS: Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. CONCLUSION: This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

In vitro killing of multidrug/extensively drug-resistant Pseudomonas aeruginosa by fosfomycin alone or in combination with antipseudomonal antibiotics.

Pseudomonas aeruginosa is a leading cause of nosocomial infections. Given the constant rise in resistance, adequate therapy is increasingly demanding. Fosfomycin recently became an appealing treatment option of bacterial infections due to multidrug-resistant bacteria (MDR). So far, fosfomycin synergy with other antibiotics has been assessed in studies, but only a limited number focused on MDR P. aeruginosa and on the effect of these combinations on the duration of the postantibiotic effect (PAE). We investigated synergy of fosfomycin with an array of antipseudomonal antibiotics using gradient diffusion strip cross method and time-kill method, and their effect on the duration of PAE against 51 variously resistant P. aeruginosa isolates. The highest rate of synergy was observed for combination with ceftazidime (23.4%) and gentamicin (19.1%). The PAE of antibiotic combinations was superior to that of the drugs alone. Our findings indicate that fosfomycin combination therapy may be a valuable treatment alternative.

IMPACT OF AIR TRANSPORT ON BLOOD SAMPLE QUALITY.

The aim of this study was to establish the impact of air transport on blood samples packaged with and without cooling elements and effect of outdoor temperature on sample quality. Venous samples from 38 blood donors in winter and 36 in summer were tested for hemolysis and complete blood count. One tube per subject was kept in controlled conditions at +4 °C. Two sets of tubes were sent by plane from Zagreb to Brussels, one with and one without cooling elements, and another two sets were sent to London following the same principle. Packages with cooling elements were stored in controlled warehousing conditions at airports (+2 °C to +8 °C), whereas packages without cooling elements were stored in ambient warehouse conditions. Data loggers were used for temperature monitoring. Our research revealed statistically significant differences in several hematologic parameters when comparing the samples stored in controlled laboratory conditions and those transported by plane. These differences were more pronounced in the samples transported during the summer. Transport conditions without cooling elements had additional negative impact on the sample quality. Transport of samples using cooling elements and controlled warehousing conditions at airports are sometimes not sufficient to maintain laboratory storage conditions.

Poly(ε-caprolactone) Titanium Dioxide and Cefuroxime Antimicrobial Scaffolds for Cultivation of Human Limbal Stem Cells.

Limbal Stem Cell Deficiency (LSCD) is a very serious and painful disease that often results in impaired vision. Cultivation of limbal stem cells for clinical application is usually performed on carriers such as amniotic membrane or surgical fibrin gel. Transplantation of these grafts is associated with the risk of local postoperative infection that can destroy the graft and devoid therapeutic benefit. For this reason, electrospun scaffolds are good alternatives, as proven to mimic the natural cells surroundings, while their fabrication technique is versatile with regard to polymer functionalization and scaffolds architecture. This study considers the development of poly(ε-caprolactone) (PCL) immune-compatible and biodegradable electrospun scaffolds, comprising cefuroxime (CF) or titanium dioxide (TiO2) active components, that provide both bactericidal activity against eye infections and support of limbal stem cells growth in vitro. The PCL/CF scaffolds were prepared by blend electrospinning, while functionalization with the TiO2 particles was performed by ultrasonic post-processing treatment. The fabricated scaffolds were evaluated in regard to their physical structure, wetting ability, static and dynamic mechanical behaviour, antimicrobial efficiency and drug release, through scanning electron microscopy, water contact angle measurement, tensile testing and dynamic mechanical analysis, antimicrobial tests and UV-Vis spectroscopy, respectively. Human limbal stem cells, isolated from surgical remains of human cadaveric cornea, were cultured on the PCL/CF and PCL/TiO2 scaffolds and further identified through immunocytochemistry in terms of cell type thus were stained against p63 marker for limbal stem cells, a nuclear transcription factor and cytokeratin 3 (CK3), a corneal epithelial differentiation marker. The electrospun PCL/CF and PCL/TiO2 successfully supported the adhesion, proliferation and differentiation of the cultivated limbal cells and provided the antimicrobial effect against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans.