Omadacycline drug susceptibility testing for non-tuberculous mycobacteria using oxyrase to overcome challenges with drug degradation.

BACKGROUND: Drug susceptibility testing (DST) protocol of omadacycline against non-tuberculous mycobacteria has not yet been established. We developed a method to accurately determine MIC omadacycline MIC against Mycobacterium abscessus (Mab), Mycobacterium avium-complex (MAC), and Mycobacterium kansasii (Mkn). METHODS: First, we identified the oxyrase concentration not affecting Mab, MAC, and Mkn growth followed by omadacycline MIC experiments with and without oxyrase using reference and clinical strains. RESULTS: Oxyrase 0.5 % (v/v) stabilized omadacycline in the culture medium. The median omadacycline MIC was 1 mg/L for Mab and 8 mg/L for Mkn. For MAC, the median omadacycline MIC was 2 mg/L for M. avium, 256 mg/L for M. intracellulare, and 4 mg/L for M. chimaera (p < 0.0001). Wilcoxon matched-pairs signed rank test revealed statistically lower MICs with oxyrase for all MAC subspecies (p < 0.0001), all Mab subspecies (p < 0.0001), and Mkn (p = 0.0002). The decrease in MICs with oxyrase was 17/18 of Mab, 14/19 of Mkn, 8/8 of M. avium, 4/5 M. chimera, but only 11/18 of M. intracellulare (p < 0.013). CONCLUSION: Use of 0.5 % oxyrase could be a potential solution to reliable and reproducible omadacycline MIC of Mab. However, oxyrase demonstrated a variable effect in reducing MICs against MAC and Mkn.

Exploring the role of E.coli derived enzyme, Oxyrase, as an oxygen scavenger to improve the cryotolerance of spermatozoa of Sahiwal bull.

The current study intended to optimize the concentration of Oxyrase in the semen dilutor and to evaluate its effect on freezability of spermatozoa of Sahiwal bulls. Supplementation of Oxyrase at 0.125 IU/mL concentration significantly reduced dissolved oxygen (DO) in the dilutor to 4 ppm in 16-18 min at 35 °C. For supplementation studies, a total of 24 ejaculates were categorized into poor and good ejaculates categories (n = 12 each) based on their initial progressive motility. Each ejaculate was further divided into two aliquotes. The first aliquote was diluted with tris-egg yolk extender without Oxyrase (control group) whereas, in the treatment group, Oxyrase was supplemented at the concentration of 0.125 IU/mL of extender. The parameters evaluated include cholesterol and plasma membrane phospholipids (PMP) at fresh, while IPM, acrosomal and plasma membrane integrity, cholesterol, PMP and oxidative stress parameters like lipid peroxidation (LPO), total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated at pre-freeze and post-thaw stages. The IPM and acrosomal intactness were higher (p < 0.05) in treatment group at post-thaw stage in good ejaculates. Oxyrase supplementation resulted in lower (p < 0.05) cholesterol leakage in both categories and lower (p < 0.05) LPO in good ejaculates at post-thaw stage. No statistical difference in ROS was observed between control and treatment groups at all stages whereas, level of TAC was higher (p < 0.05) in the treatment group compared to control group at post-thaw stage of both categories. Therefore, Oxyrase as an oxygen scavenging agent could preserve the post-thaw quality of Sahiwal bull spermatozoa.

Escherichia coli membrane-derived oxygen-reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation.

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

Effect of combination of Oxyrase and sodium thioglycolate on growth of Clostridium perfringens from spores under aerobic incubation.

Clostridium perfringens is a strictly anaerobic pathogen that requires absence of oxygen for its growth in laboratory experiments, which is usually attained by using an anaerobic chamber or anaerobic jars. However, it has been demonstrated that C. perfringens may survive for short periods of times due to its adaptive response to O2. Therefore, the objective of this study was to explore the application of Oxyrase (OX) and sodium thioglycolate (ST) as oxygen scavengers, used alone or in combination, for observation of the growth of C. perfringens under aerobic incubation. The growth of C. perfringens from spores in Schaedler Anaerobe Agar containing different levels and combinations of OX and ST was observed at temperatures between 20 and 50 °C. The kinetic parameters, including lag time, specific growth rate, and maximum cell concentrations in the stationary phase, were determined. The results indicated that ST at concentrations of 0.025 and 0.05% (w/w), although allowing eventual growth of C. perfringens, prolonged its lag times, while OX at 1.5% only allowed growth at a lower growth rate in comparison to anaerobic incubation. OX at 3% enhanced the growth of C. perfringens at temperatures between 30 and 50 °C, while higher levels of OX were needed in the medium to support the growth of C. perfringens during storage at 25 °C (>6% OX) and 20 °C (>9% OX), due to the effect of temperature on enzyme activity. No significant difference was found in the kinetic parameters of C. perfringens incubated aerobically with OX and the control (without OX or ST) in an anaerobic chamber. Therefore, OX at appropriate concentrations may allow the observation of the growth of C. perfringens under aerobic incubation conditions without the need of an anaerobic device.

Artificially decreased dissolved oxygen increases the persistence of Trichomonas gallinae in water.

Water containing organic material has been shown to increase the persistence of the avian pathogenic protozoa, Trichomonas gallinae. We hypothesized that the decrease in dissolved oxygen due to microbes in the organic material could increase persistence of the microaerophilic trichomonads. Using simulated birdbaths, we determined 1) the levels of dissolved oxygen in distilled water with various amounts of organic material, 2) the concentration of the oxygen-scavenging enzyme Oxyrase® needed to achieve the dissolved oxygen levels obtained in organic material contaminated water, and finally, 3) the persistence of two T. gallinae isolates in Oxyrase®-supplemented water. An average of 9.6% dissolved oxygen was obtained with the addition of 15 g organic material to 500 ml of distilled water, whereas organic material-free water had 86.2% dissolved oxygen. The addition of 0.5% and 1.0% (vol/vol) Oxyrase® to organic material-free water yielded dissolved oxygen of 18.6% and 6.9%, respectively. Using 0.5% and 1.0% concentrations of Oxyrase®, we evaluated the persistence of two trichomonad isolates by inoculating ∼1 million trichomonads into 500 ml distilled water in triplicate. At various time-points, 0.5 ml aliquots of trichomonad-inoculated water were obtained and placed into Hollander Fluid media, incubated at 37 °C, and read by light microscopy every other day for 5 days. In our 1% Oxyrase® treatments, the longest recorded persistence of broad-winged hawk 1 increased from the previously reported 4hrs to 30hrs and Cooper's hawk 4 from 16hrs to 30hrs. These results indicate that the mechanism for organic material-mediated trichomonad persistence is associated with decreased dissolved oxygen, further demonstrating the importance of keeping birdbaths free of organic debris to discourage trichomonad persistence.

Low-Cost Method Generating In Situ Anaerobic Conditions on a 96-Well Plate for Microbial Fermentation in Food Research.

Commercial tools and instruments have been developed for a screening study of microbial fermentation, but they are expensive and mostly confined to aerobic fermentation only. There is little development on the generation of anaerobic conditions directly on a 96-well plate. This report proposed a simple and versatile microbial fermentation system known as OVAMO that makes use of Oxyrase, vacuum, and mineral oil to generate an in situ anaerobic environment on a 96-well plate for at least 48 h. The practicality of OVAMO in anaerobic fermentation experiments used for functional food research was validated by a prebiotic screening study of different carbohydrates by Bifidobacterium longum subsp. infantis. The OVAMO system provides a less expensive but effective way to conduct a microbial fermentation screening study that requires anaerobic conditions without the need for atmospheric control by external devices.