Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers.

Novel Platform for Regulation of Extracellular Vesicles and Metabolites Secretion from Cells Using a Multi-Linkable Horizontal Co-Culture Plate

Microfluidics is applied in biotechnology research via the creation of microfluidic channels and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example is the cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts have been used for years to study cell-to-cell communication. These systems generally have a bucket-in-bucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However, VTCPs have several disadvantages, such as the inability to simultaneously observe samples in both containers and the inability of cell-to-cell communication through the filters at high cell densities. In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome these disadvantages and confirm its performance. In addition, we clarified the migration characteristics of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that less material is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer was found to be twice as high when using HTCP. Other merits include control of the degree of co-culture via the placement of cells. We believe that this novel HTCP container will facilitate research on cell-to-cell communication in various fields.

Comparative study of antibiofilm activity of Lime juice and Lithium dioxide nanoparticles against E.coli isolated from local made cheese

Thirty specimens of fresh white cheese, in different markets at different cities of Iraq were analyzed microbiologically. Isolates of E.coli that have been collected from the samples of cheese, were investigated. Capacity for biofilm producing was demonstrated by two method, Tissue culture plate method (TCP) and agar (CRA). After that, antibiofilm activity of lime extract and LiO2NPs was studied as each one of them alone and then the synergistic effect was done by TCP method. The results showed that all E.coli isolates produce biofilm but in different degrees. The results also displayed that Lime extract and LiO2NPs had antibiofilm effect against E.coli when used alone and when the combination done between each one of these materials. In conclusion, it was observed that the specimens of fresh white cheese included in this study contained microbial contamination at a health-threatening level but elimination of this contamination can be done by using lime extract and LiO2NPs.

Biofilm formation by bacteria isolated from intensive care units of a tertiary care hospital, with special relevance to its risk factors

Background: The purpose of this study was to detect biofilm formation by bacterial isolates from patients with device associated infection admitted in intensive care units (ICUs), to compare the three methods used for detection of bioiflm, to compare the antimicrobial susceptibility pattern of the biofilm producers with the non-producers and to study the risk factors associated with biofilm formation.Methods: A total of 115 bacterial isolates from patients with device associated infection admitted in different ICU for a period of one year was included in the study. These clinical isolates were detected for biofilm formation by tissue culture plate method, tube method and Congo red agar method. Kirby-Bauer disc diffusion method of antibiotic susceptibility was performed on all isolates.Results: Out of the 115 bacterial isolates, 71 were biofilm producers. Tissue culture plate method detected the maximum number of biofilm producers (61.7%). The maximum number of biofilm producers were isolated from tracheal aspirate and endotracheal tubes (52.1%) followed by blood (17%) and urine (12.6%) respectively. The predominant biofilm producing isolates were Klebsiella pneumoniae (39.4%), Staphylococcus aureus (19.7%) and Pseudomonas aeruginosa (16.9%). Multi drug resistance among the biofilm producers was significantly higher than the non-biofilm producers (p value=0.0125). The risk of biofilm formation was seen to increase with the increase in duration of hospital stay (p value=0.0092, statistically very significant).Conclusions: From this study it was found that a high degree of biofilm producers were isolated from patients on indwelling devices. Tissue culture plate was found to be the most accurate method. The degree of multidrug resistance among the bioiflm producers was significantly higher than the non-producers.

Effects of Non-thermal Ultrasound on a Fibroblast Monolayer Culture: Influence of Pulse Number and Pulse Repetition Frequency

Despite the use of therapeutic ultrasound in the treatment of soft tissue pathologies, there remains some controversy regarding its efficacy. In order to develop new treatment protocols, it is a common practice to carry out in vitro studies in cell cultures before conducting animal tests. The lack of reproducibility of the experimental results observed in the literature concerning in vitro experiments motivated us to establish a methodology for characterizing the acoustic field in culture plate wells. In this work, such acoustic fields are fully characterized in a real experimental configuration, with the transducer being placed in contact with the surface of a standard 12-well culture plate. To study the non-thermal effects of ultrasound on fibroblasts, two different treatment protocols are proposed: long pulse (200 cycles) signals, which give rise to a standing wave in the well with the presence of cavitation (ISPTP max = 19.25 W/cm2), and a short pulse (five cycles) of high acoustic pressure, which produces a number of echoes in the cavity (ISPTP = 33.1 W/cm2, with Pmax = 1.01 MPa). The influence of the acoustic intensity, the number of pulses, and the pulse repetition frequency was studied. We further analyzed the correlation of these acoustic parameters with cell viability, population, occupied surface, and cell morphology. Lytic effects when cavitation was present, as well as mechanotransduction reactions, were observed.

Phenotypic and genotypic characterization of biofilm producing clinical coagulase negative staphylococci from Nepal and their antibiotic susceptibility pattern

Background Coagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns. Methods A total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes. Results Among all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates. Conclusion The study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.

Comparison of Apical and Basolateral Cu Treatment for Iron-related Gene Regulation During Deferoxamine Induced Iron Deficiency Anemia

Background: Intestinal copper transporter (Atp7a) mutant brindled mice with systemic Cu deficiency had elevated Cu levels in enterocyte cells without any perturbation of iron regulating genes, suggesting that blood Cu level might be important for intestinal iron homeostasis during iron deficiency anemia (IDA). We hypothesized that the blood Cu level and polarization (apical and basolateral) of enterocyte cells might be important regulators for the compensatory response on the regulation of genes in enterocyte cells during IDA. Methods: We grew Caco-2 cells on a bicameral cell culture plate to mimic the human intestine system and on a regular tissue culture plate. IDA was induced by Deferoxamine (DFO). The cells were treated with Cu and Cu with Fe following mRNA expressions of DMT1, FPN, TFR, and ANKRD37 were analyzed. Results: Our main finding was that basolateral treatment of Cu significantly reduced mRNA expressions of iron-regulated genes, including DMT1, FPN, TFR, and ANKRD37, compared to DFO treated and DFO with apical Cu treated groups in both bicameral and regular tissue culture plates. Conclusions: Cu level in the basolateral side of Caco-2 cells significantly influenced the intracellular gene regulation in DFO induced iron-deficient condition, and polarization of the cells might be important factor gene regulation in enterocyte cells.

Correlation between antimicrobial resistance and biofilm formation capability among Klebsiella pneumoniae strains isolated from hospitalized patients in Iran

Background Klebsiella pneumoniae is a common cause of nosocomial infections. Antibiotic resistance and ability to form biofilm, as two key virulence factors of K. pneumoniae, are involved in the persistence of infections. The purpose of this study was to investigate the correlation between antimicrobial resistance and biofilm formation capability among K. pneumoniae strains isolated from hospitalized patients in Iran. Methods Over a 10-month period, a total of 100 non-duplicate K. pneumoniae strains were collected. Antibiotic susceptibility was determined by Kirby–Bauer disk diffusion method according to CLSI. Biofilm production was assessed by tissue culture plate method. Finally, polymerase chain reaction was conducted to detect four families of carbapenemase: blaIMP, blaVIM, blaNDM, blaOXA−48; biofilm formation associated genes: treC, wza, luxS; and K. pneumoniae confirming gene: rpoB. Results Most of the isolates were resistant to trimethoprim-sulfamethoxazole (52 %), cefotaxime (51 %), cefepime (43 %), and ceftriaxone (43 %). Among all the 100 isolates, 67 were multidrug-resistant (MDR), and 11 were extensively drug-resistant (XDR). The prevalence of the blaVIM, blaIMP, blaNDM, and blaOXA−48 genes were 7 , 11 , 5 , and 28 %, respectively. The results of biofilm formation in the tissue culture plate assay indicated that 75 (75 %) strains could produce biofilm and only 25 (25 %) isolates were not able to form biofilm. Among these isolates, 25 % formed fully established biofilms, 19 % were categorized as moderately biofilm-producing, 31 % formed weak biofilms, and 25 % were non-biofilm-producers. The antimicrobial resistance among biofilm former strains was found to be significantly higher than that of non-biofilm former strains (p < 0.05). Molecular distribution of biofilm formation genes revealed that 98 , 96 , and 34 % of the isolates carried luxS, treC, and wza genes, respectively. Conclusions The rise of antibiotic resistance among biofilm-producer strains demonstrates a serious concern about limited treatment options in the hospital settings. All of the data suggest that fundamental actions and introduction of novel strategies for controlling of K. pneumoniae biofilm-related infections is essential.