Absence Of Microbial Contamination Research Articles

In vitro study on antidiabetic and antihypertensive activities of ethanolic extract of propolis of Indonesian stingless bee Tetragonula sapiens

The stingless bee, Tetragonula sapiens, is one of the species producing propolis in Indonesia. To date, there is still limited information about the standard characteristics and biological activity of propolis. This study investigated the standardized extract of Indonesian propolis in terms of its potency for controlling diabetes and high blood pressure in vitro, in accordance with its standard parameters. Three types of extracts; i.e., ethanolic extract of propolis (EEP), spray-dry propolis without wax (SDPNW), and spray-dry propolis with wax (SDPW); were prepared from T. sapiens stingless bees collected in South Sulawesi, Indonesia. The ethanolic extractable, loss on drying, ash, and water content parameters of EEP, SDPNW, and SDPW met the natural extract requirement. There is no significant value for their residual solvent levels (<0.54%), heavy metals (the absence of As, Cd, Pb, and Hg contamination), or the absence of microbial contamination. ACE inhibition activity on captopril, EEP, SDPNW, and SDPW had IC50 values of 5.97, 115.06, 112.93, and 116.70 ppm, respectively. While the IC50 values of acarbose, SDPNW, EEP, and SDPW for antidiabetic activity of α-glucosidase inhibition were 53.34, 56.48, 60.09, and 68.28 ppm, respectively. The TFC and TPC content of SDPNW were higher than EEP and SDPW, with values of 1.59% and 1.93%, respectively. It can be said that Indonesian propolis extract meets the standards for an extract and has the potential to reduce diabetic and hypertensive diseases. Further preclinical and clinical studies are needed to develop standardized propolis extract as an anti-diabetic and antihypertensive agent.

Design and Characterization of a Cheese Spread Incorporating Osmundea pinnatifida Extract.

Marine algae have been emerging as natural sources of bioactive compounds, such as soluble dietary fibers and peptides, presenting special interest as ingredients for functional foods. This study developed a cheese spread incorporating red seaweed Osmundea pinnatifida extract and subsequently characterized it in terms of nutritional, pH, and microbiological parameters and bioactivities including prebiotic, antidiabetic, antihypertensive, and antioxidant activities. This food was produced through incorporation of O. pinnatifida extract (3%), obtained via enzymatic extraction Viscozyme L in a matrix containing whey cheese (75%) and Greek-type yoghurt (22%). The product was then subjected to thermal processing and subsequently stored for 21 days at 4 °C. During storage, this food showed a high pH stability (variations lower than 0.2 units), the absence of microbial contamination and all tested bioactivities at the sampling timepoints 0 and 21 days. Indeed, it exerted prebiotic effects under Lactobacillus acidophilus LA-5® and Bifidobacterium animalis subsp. lactis BB-12®, increasing their viability to around 4 and 0.5 log CFU/g, respectively. In addition, it displayed antidiabetic (α-glucosidase inhibition: 5-9%), antihypertensive (ACE inhibition: 50-57%), and antioxidant (ABTS: 13-15%; DPPH: 3-5%; hydroxyl radical: 60-76%) activities. In summary, the cheese spread produced may be considered an innovative food with high potential to contribute toward healthier status and well-being of populations.

National Collection of Pathogenic Viruses: A Repository for Well Characterised and Authenticated Viruses

The National Collection of Pathogenic Viruses (NCPV) is one of four repositories making up Public Health England Culture Collections. It was established two decades ago to offer safe and secure facilities for storage of patents and deposits as well as to supply standardised virus products to the wider scientific community. Within the collection are strains of both medical and veterinary importance, with representation from a wide variety of virus families, classified as Hazard Groups 2, 3 and 4 by the Advisory Committee for Dangerous Pathogens (ACDP). These include arboviruses such as Dengue, Yellow-fever, Oropouche and Sandfly fever Naples. Throughout the year, the collection receives virus deposits. The depositing process is free and simple. The quality of every virus material received in the collection is assessed through a series of tests to confirm viability and absence of microbial contamination (including mycoplasmas). Virus identity is confirmed by nucleic acid profiling. Once standards are met, the virus products are made available to the wider scientific community. In addition to facilitating access to viruses and their associated products as well as secure storage for patents, NCPV also undertakes collaborative projects and contract research/developmental work with academic and commercial partners, by providing expertise in the handling of viral pathogens. A repository for well-characterised, authenticated viruses is a useful resource for the scientific community, aiding in the understanding of circulating viral pathogens and providing access to quality-assured reference reagents for the identification and development of vaccines and therapeutics in the event of global viral outbreaks.

Comparing Physical Container Closure Integrity Test Methods and Artificial Leak Methodologies.

The sterility of drug products intended for parenteral administration is a critical quality attribute (CQA) because it serves to ensure patient safety and is thus a key requirement by health authorities. While sterility testing is a probabilistic test, the assurance of sterility is a holistic concept including adequate design of manufacturing facilities, process performance, and product design. Container closure integrity testing (CCIT) is necessary to confirm the integrity of a container closure system (CCS), until the end of a product's shelf life. The new and revised United States Pharmacopeia (USP) General Chapter <1207> is a comprehensive guidance on CCI. Nevertheless, practical considerations including the choice of CCIT methods, the acceptance criteria, or the positive control samples (artificial leaks) must be addressed by the pharmaceutical manufacturer.This study is the first to provide a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA), and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).The results from these experiments provide comprehensive data to allow a direct comparison of the capabilities of the individual methods. The results confirmed that the He leak detection method, which is considered the "gold-standard" for pCCIT regarding method sensitivity, indeed demonstrates the highest detection sensitivity (lowest detection limit). In comparison to the dye ingress method, HSA and vacuum decay also demonstrated better detection sensitivity in our study.Capillary leaks with orifice diameter (capillary leak with flow according to an ideal orifice) and micro holes yielded similar leak rates, whereas capillaries with nominal diameters yielded significantly lower leak rates. In conclusion, method sensitivity cannot be compared by means of a leak diameter, but requires the consideration of multiple impacting factors (e.g., path length, uniformity).LAY ABSTRACT: Sterility of drug products intended for parenteral administration is a critical quality attribute to ensure patient's safety and is thus a key requirement by health authorities. The absence of microbial contamination must be demonstrated by container closure integrity (CCI) of the container closure system (CCS). Currently, the revised United States Pharmacopeia (USP) General Chapter <1207> provides the most extensive guidance on how CCI should be assessed. Nevertheless, practical considerations on the choice of an appropriate CCIT method, artificial leaks or the choice of an acceptance criteria are lacking and must be addressed by the pharmaceutical manufacturer.This study provides a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA) and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).

Decoronation followed by dental implants placement: fundamentals, applications and explanations.

ABSTRACTDental arches areas with teeth presenting dentoalveolar ankylosis and replacement root resorption can be considered as presenting normal bone, in full physiological remodeling process; and osseointegrated implants can be successfully placed. Bone remodeling will promote osseointegration, regardless of presenting ankylosis and/or replacement root resorption. After 1 to 10 years, all dental tissues will have been replaced by bone. The site, angulation and ideal positioning in the space to place the implant should be dictated exclusively by the clinical convenience, associated with previous planning. One of the advantages of decoronation followed by dental implants placement in ankylosed teeth with replacement resorption is the maintenance of bone volume in the region, both vertical and horizontal. If possible, the buccal part of the root, even if thin, should be preserved in the preparation of the cavity for the implant, as this will maintain gingival tissues looking fully normal for long periods. In the selection of cases for decoronation, the absence of microbial contamination in the region - represented by chronic periapical lesions, presence of fistula, old unconsolidated root fractures and active advanced periodontal disease - is important. Such situations are contraindications to decoronation. However, the occurrence of dentoalveolar ankylosis and replacement resorption without contamination should neither change the planning for implant installation, nor the criteria for choosing the type and brand of dental implant to be used. Failure to decoronate and use dental implants has never been reported.

Humidifiers for oxygen therapy: what risk for reusable and disposable devices?

Nosocomial pneumonia accounts for the vast majority of healthcare-associated infections (HAI). Although numerous medical devices have been discussed as potential vehicles for microorganisms, very little is known about the role played by oxygen humidifiers as potential sources of nosocomial pathogens. The purpose of this research was to evaluate the safety of the reuse of humidifiers by analysing the rate of microbial contamination in reusable and disposable oxygen humidifiers used during therapy, and then discuss their potential role in the transmission of respiratory pathogens. Water samples from reusable and disposable oxygen humidifiers were collected from different wards of the University Hospital of Messina, Italy, where nosocomial pneumonia has a higher incidence rate due to the "critical" clinical conditions of inpatients. In particular, we monitored the Internal Medicine and Pulmonology wards for the medical area; the General Surgery and Thoracic and Cardiovascular Surgery wards for the surgical area and the Intensive Care Unit and Neonatal Intensive Care Unit for the emergency area. The samples were always collected after a period of 5 days from initial use for both types of humidifiers. Samples were processed using standard bacteriological techniques and microbial colonies were identified using manual and automated methods. High rates of microbial contamination were observed in samples from reusable oxygen humidifiers employed in medical (83%), surgical (77%) and emergency (50%) areas. The most relevant pathogens were Pseudomonas aeruginosa, amongst the Gram-negative bacteria, and Staphylococcus aureus, amongst the Gram-positive bacteria. Other pathogens were detected in lower percentage. The disposable oxygen humidifier samples showed no contamination. This research presents evidence of the high rate and type of microbial contamination of reusable humidifiers employed for oxygen therapy. These devices may thus be involved in the transmission of potential pathogens. It could be important, for the prevention of nosocomial pneumonia, to replace them with singleuse humidifiers for which the absence of microbial contamination has been confirmed.

Investigation of Endophytic Bacterial Community in Supposedly Axenic Cultures of Pineapple and Orchids with Evidence on Abundant Intracellular Bacteria.

Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."

Effects of reclaimed wastewater irrigation on olive (Olea europaea L. cv. ‘Koroneiki’) trees

Irrigation of olive trees with treated wastewaters in arid and semi-arid regions is becoming a necessary alternative to addressing issues of water shortages. The irrigation requires a careful monitoring of soil and plants for a range of parameters including salts, nutrients, micro-elements, heavy metals, toxic pollutants, and pathogens. A 3-years monitoring of the above parameters in soil and leaves of olive trees in Crete, Greece was conducted using trees that were irrigated with three types of water: secondary treated wastewater (STW), tertiary treated wastewater (TTW), and tap water (TW). Plant growth was similar for all irrigation sources as indicated by measured trunk diameter, plant height, and photosynthetic activity. In addition, no significant differences in mineral leaf content were observed. Accumulation of nutrients (mainly P and K) and salts (Na) in soils 2 years after the application of both types of treated wastewater were observed in comparison to tap water. Magnesium, calcium, and boron concentrations in treated wastewaters were higher than TW. However no effects on soil concentrations were reported. Concentration of polycyclic aromatic hydrocarbons in soil ranged from 349.0μgkg−1 to 395.4μgkg−1 values not significantly different between the examined water sources. Finally, E. coli mean content in STW was 3580±800MPN 100mL−1; however, the absence of microbial contamination was detected in leaves and fruits of olive trees. The results of this study suggest that STW and TTW can be applied successfully for the safe irrigation of the olive groves.

Blood Glucose-lowering Effect of T. procumbens L.: A Pilot Clinical Study in Individuals with Type 2 Diabetes.

Traditional knowledge, in vitro studies, and studies using animal models suggest that Tridax procumbens L. exhibits blood glucose-lowering properties and antiinflammatory effects. In this study, we evaluated the blood glucose-lowering effect of T. procumbens supplementation in individuals with type 2 diabetes. An extract (asava) of T. procumbens L. was prepared following Ayurveda guidelines. Chemical and microbial analyses indicated presence of phenolics, flavonoids, and carotenoids, and absence of microbial contamination, aflatoxins, heavy metals, and pesticide residues. A chemical fingerprint of T. procumbens L. asava, developed using Ultra high pressure liquid chromatography/electron spray ionization-mass spectrometry (UPLC/ESI-MS) in negative mode, suggest the presence of several compounds including polyphenols. T. procumbens asava demonstrated strong total antioxidant capacity, Fe3+ reducing potential, Fe2+ chelation, H2 O2 scavenging activity, and inhibition of lipid peroxidation. We recruited 20 type 2 diabetic individuals from Kolhapur, India. Participants received 15 mL of T. procumbens asava, twice daily, for 4 weeks, while continuing their prescribed antidiabetic medications. Fasting blood glucose decreased by 11% in men (p < 0.01) and 20% in women (p < 0.05), and post-prandial blood glucose concentrations were lowered by 26% in men (p < 0.001) and 29% in women (p < 0.001) following 4 weeks of asava supplementation. No adverse events or side effects were reported. This is the first clinical study demonstrating a significant blood glucose-lowering effect of T. procumbens asava in type 2 diabetes. Copyright © 2015 John Wiley & Sons, Ltd.

Considerations in the sterile manufacture of polymeric microneedle arrays.

We describe, for the first time, considerations in the sterile manufacture of polymeric microneedle arrays. Microneedles (MN) made from dissolving polymeric matrices and loaded with the model drugs ovalbumin (OVA) and ibuprofen sodium and hydrogel-forming MN composed of "super-swelling" polymers and their corresponding lyophilised wafer drug reservoirs loaded with OVA and ibuprofen sodium were prepared aseptically or sterilised using commonly employed sterilisation techniques. Moist and dry heat sterilisation, understandably, damaged all devices, leaving aseptic production and gamma sterilisation as the only viable options. No measureable bioburden was detected in any of the prepared devices, and endotoxin levels were always below the US Food & Drug Administration limits (20 endotoxin units/device). Hydrogel-forming MN were unaffected by gamma irradiation (25 kGy) in terms of their physical properties or capabilities in delivering OVA and ibuprofen sodium across excised neonatal porcine skin in vitro. However, OVA content in dissolving MN (down from approximately 101.1 % recovery to approximately 58.3 % recovery) and lyophilised wafer-type drug reservoirs (down from approximately 99.7 % recovery to approximately 60.1 % recovery) was significantly reduced by gamma irradiation, while the skin permeation profile of ibuprofen sodium from gamma-irradiated dissolving MN was markedly different from their non-irradiated counterparts. It is clear that MN poses a very low risk to human health when used appropriately, as evidenced here by low endotoxin levels and absence of microbial contamination. However, if guarantees of absolute sterility of MN products are ultimately required by regulatory authorities, it will be necessary to investigate the effect of lower gamma doses on dissolving MN loaded with active pharmaceutical ingredients and lyophilised wafers loaded with biomolecules in order to avoid the expense and inconvenience of aseptic processing.

Stability of multidose, preserved formulation epoetin alfa in syringes for three and six weeks.

The integrity and biological activity of multidose, preserved formulation epoetin alfa stored in syringes at 2-8 degrees C were studied. Three independent 1.0-mL hubless syringes of epoetin alfa 20,000 units/mL were aseptically prepared and refrigerated for three and six weeks (a total of six syringes). Protein integrity was assayed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein detection. Biological activity was determined through a cell-based proliferation assay. The presence or absence of microbial contamination was observed after a one-week culture. A multidose, preserved formulation of epoetin alfa that was opened only at the time of assay served as the reference standard. SDS-PAGE silver-stained gels and immunoblots demonstrated no evidence of erythropoietin degradation after three and six weeks of storage when compared with the reference standard. In addition, SDS-PAGE, immunoblotting, and direct glycoprotein detection found that protein glycosylation was unaffected by the storage. Student's t test detected no significant difference between stored samples and the reference standard in biological activity (p > 0.05). A culture of epoetin alfa in bacterial and eukaryotic cell growth media showed no evidence of contamination. The results suggest that epoetin alfa can be dispensed to patients in prefilled syringes every four to six weeks to coincide with their peritoneal dialysis schedule. The integrity and biological activity of 20,000 units/mL epoetin alfa in prefilled syringes remain intact after three and six weeks when stored at 2-8 degrees C.

Germfree animals and technics in surgical research

Germfree animals have been reared to a size, weight, and age permitting the performance of major surgical procedures and the pursuit of a variety of surgical research problems. Germfree dogs have been maintained in the isolator system through three generations, indicating that life, reproduction, and growth are all possible in the absence of microbial contamination. The value of the germfree approach to surgical problems has been utilized in studies of a variety of gastrointestinal problems, shock, cancer, immunology, burns, wound healing, and in direct patient application. Patients have been maintained in isolator environments for prevention of infection, for operative procedures, for treatment of extensive burns, and for management of immune-suppressed individuals. We conclude that germfree animals and germfree technics provide a valuable addition to the armamentarium of the surgeon in both research and clinical applications.

The replication of two California serogroup viruses in a cell line from the mosquito Aedes triseriatus (Diptera: Culicidae).

An Aedes triseriatus cell line was established from neonatal larval tissue. In the 60th passage, the cells have been characterized as to growth, chromosome number, absence of microbial contamination, and absence of La Crosse encephalitis virus (LAC) and other vertebrate viruses as determined by passage in suckling mice and electron microscopy. Isozyme analysis using polyacrylamide gel electrophoresis of lysed cell suspensions and suspensions of Aedes triseriatus larvae revealed that 4 enzyme phenotypes (isocitrate dehydrogenase, malic enzyme, phosphoglucoisomerase, and phosphoglucomutase) were identical. The Aedes triseriatus cells grow in plastic Falcon flasks in a medium (MM/VP12) composed of equal volumes of Mitsuhashi-Maramorosch and Varma-Pudney media with fetal bovine serum and antibiotics. The growth characteristics of 2 California serogroup viruses (La Crosse and trivittatus) in these cells are described