Bacterial Sterility Research Articles

Problems of Assessment of Live Bacterial Vaccine Sterility

Objective of the study - analysis of standard indicators and methods, utilized for determination of presence of contaminating microorganisms in live bacterial vaccines. Materials and methods . We used the data from the State Pharmacopeia of the USSR, 9th-11th editions; State Pharmacopeia of the Russian Federation, 12th-14th editions; as well as regulatory documentation/manufacturer’s pharmacopoeial monographs for 9 items of live vaccines. Results and discussion . Taking into account the specificity of live vaccines, the process of their manufacturing and quality control must target elimination of the possibility of contamination with microorganisms that differ from production strains. It is established that currently there is no unified terminology for determining the indicator in the Russian Federation, as well as clear-cut criteria for interpretation of test results for quality assessment of live bacterial vaccines when testing sterility/contamination with foreign bacteria and fungi. In compliance with the requirements of the current RF Pharmacopeia editions, detection of contamination in live vaccines for parenteral administration should be carried out using various methods and assessment criteria (General Pharmacopeia Monograph (GPM) “Sterility” and GPM “Microbiological purity”). Performed investigations have revealed the necessity to enhance the regulatory framework in regard to detection of contamination with foreign bacteria and fungi. It is advised to use unified nomination of the indicator, specifically, “Absence of foreign bacteria and fungi” in the normative documents. Given are the recommendations on improvement of methods and requirements to the assessment of live bacterial vaccine contamination. Developed proposals on harmonization of the quality assessment requirements of vaccines containing other live microorganisms can be used for drawing up corresponding normative-regulatory documents (GPM, Pharmacopeia monograph, regulatory documentation et al.).

Essential oil nanoemulsions: antibacterial activity in contaminated fruit juices

SummaryMany human acid tolerant bacterial and fungal pathogens can be transmitted through the consumption of the contaminated fruit juices. We aim to formulate essential oil nanoemulsions (basil, black seed, turmeric, clove & cinnamon), determine their ability to clear contamination by food borne bacterial pathogens from fruit juices. The antibacterial activity of the optimised formulations was tested in the fruit juices against bacterial pathogens causing gastrointestinal tract infections. The minimum bactericidal concentration (MBC) of clove emulsions ranged from 15.6 to 25 μL mL−1. Cinnamon oil emulsion had an MBC ranging between 15 and 31 μL mL−1. At MBC, cinnamon oil emulsions caused a 6log10 decrease in viable counts by 8 h and maintained the sterility of fruit juices for 7 days at ambient temperature. Thus, clove and cinnamon microemulsions can be used as juice additives to control food borne bacterial pathogens and maintain the bacterial sterility of fruit juices.

Nouveau test ultrarapide pour la détection de bactéries

Introduction Bacteriological testing is aimed to reduce the risk of transmission of infections. However, the detection of Bacteria by culture requires from 18 hours to 14 days and may produce erroneous results for fastidious species. The goal of this work was to design and validate a new tool for bacterial testing. Methods The test is based on the fast real-time PCR (frt PCR). The DNA extracted from samples containing internal controls are introduced into four tubes containing primers and probes for the frt PCR. The cycling program consists in 1 × at 95 °C for 10 min and 45 × (15 s at 95 °C, 8 s) at 52 °C and 10 s at 72 °C. Results The frt PCR detects 0,01 CFU/μl of Bacteria and identifies eight Genera without interferences from the environment or from fungi and with no need for melting curve analysis or additional sequencing. Discussion The frt PCR detects and quantifies Bacteria identifying and assessing the load of Staphylococci, Streptococci, Haemophilus, Pseudomonas, Enterobacteria, Acinetobacter, Propionibacteriacae and Corynebacteria. Conclusion Cultures require at least 24 hours but the new frt PCR reduces the time to 90 minutes. Larger series of samples are necessary to confirm the usefulness of this new test for routine bacterial sterility controls.

Solvent-detergent filtered (S/D-F) fresh frozen plasma and cryoprecipitate minipools prepared in a newly designed integral disposable processing bag system

Solvent-detergent (S/D) viral inactivation was recently adapted to the treatment of single plasma donations and cryoprecipitate minipools. We present here a new process and a new bag system where the S/D reagents are removed by filtration and the final products subjected to bacterial (0.2 microm) filtration. Recovered and apheresis plasma for transfusion (FFP) and cryoprecipitate minipools (400 +/- 20 mL) were subjected to double-stage S/D viral inactivation, followed by one oil extraction and a filtration on a S/D and phthalate [di(2-ethylhexyl) phthalate (DEHP)] adsorption device and a 0.2 microm filter. The initial and the final products were compared for visual appearance, blood cell count and cell markers, proteins functional activity, von Willebrand factor (VWF) multimers and protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Tri (n-butyl) phosphate (TnBP) was quantified by gas chromatography and Triton X-45 and DEHP by high-performance-liquid chromatography (HPLC). General safety tests were by 6.5 mL/kg intravenous injection in rats. The treated plasmas and cryoprecipitates were very clear and the protein content and functionality, VWF multimers and SDS-PAGE profiles were well preserved. TnBP and Triton X-45 were < 1 and <25 ppm, respectively, and DEHP (about 5 ppm) was less than it was in the starting materials. Blood cell counts and CD45, CD61 and glycophorin A markers were negative. There was no enhanced toxicity in rats. Thus, plasma and cryoprecipitate can be S/D-treated in this new CE-marked disposable integral processing system under conditions preserving protein function and integrity, removing blood cells, S/D agents and DEHP, and ensuring bacterial sterility. This process may offer one additional option to blood establishments for the production of virally inactivated plasma components.

Microbiologic Assessment of Multidose Restylane for Facial Augmentation

Microbiologic Assessment of Multidose Restylane for Facial Augmentation

Regulation of phagocyte lifespan in the lung during bacterial infection

The innate-immune response to infection is critically dependent on the antimicrobial actions of macrophages and neutrophils. Host and pathogen have evolved strategies to regulate immune-cell antimicrobial functions via alterations in cell death. Modulation of phagocyte death by bacteria is an important pathogenic mechanism. Host benefits of phagocyte apoptosis also exist, and understanding the mechanisms and consequences of apoptosis is essential before we can devise strategies to modulate this element of the innate-immune response to the host's benefit. This is of particular importance in an organ such as the lung, in which the balance between the need to recruit phagocytes to maintain bacterial sterility and the requirement to clear recruited cells from the alveolar units to preserve physiologic gas exchange must be finely tuned to ensure survival during bacterial infection. Apoptosis clearly plays a critical role in reconciling these physiological requirements.

Monocyte-derived dendritic cells: development of a cellular processor for clinical applications

Since dendritic cells (DCs) are the most professional antigen-presenting cells, (Schuler et al., 1997), increasing interest in their use in clinical approaches has been observed. (Nestle et al., 1998; Murphy G. et al., 1996). We have developed an ex vivo standardized process for the generation of dendritic-like cells (MAC-DCs) from human blood circulating monocytes. Human monocytes can differentiate into very different functional cells according to the conditions of culture, media and cytokines used. In the present study, we demonstrate that both pure monocytes and mononuclear cells differentiate into DCs when they are grown in defined medium AIM-V in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IL13 and in approved biocompatible non-adherent bags. Quality and functional controls of the immature DCs obtained rely on bacterial sterility, viability, morphology and recovery. The MAC-DCs also present an immature DC phenotype with a low expression of CD14 and CD64, and high expression of MHC-I, MHC-II and CD40. They also express B7 costimulatory molecules (CD80, CD86), CD83, and CD1a molecules. They induce strong allogenic T-cell proliferation (mixed lymphocyte reaction as well as proliferation of autologous memory T lymphocytes when incubated in the presence of recall antigens (tuberculosis, Candida albicans, and tetanus toxoid). They also show an increase in phagocytic uptake of yeast, tumour cells and debris. The global closed system which, under reproducible good medical practice (GMP) conditions, enables the production of dendritic cells of clinical quality, has been optimized ("Vac Cell Processor"). It contains all bags, connections, media, reagents, washing solutions, control antibodies, standard operating procedures, data management, traceability and help in the form of dedicated software.

The extension of 4 degrees C storage time of frozen-thawed red cells.

Blood was drawn from volunteer donors and frozen using the high glycerin, mechanical freezing procedure accepted by the United States Navy. Subsequently, the units of blood were thawed and washed. Various anticoagulants were added, and the red cells were stored in a refrigerator at 4 degrees C for periods of up to 28 days. Chemical analyses were performed periodically. These showed that the addition of the anticoagulants ACD, CPD and CPDA-1 caused the red cells to be preserved better than the currently accepted 0.9-percent NaCl, 0.2-percent glucose solution. In vivo 51Cr viability studies performed on blood stored with CPDA-1 for 14 days showed a 24-hour viability of 78.8 +/- 8.4 percent. In a subsequent study, the blood was stored for 21 days prior to freezing and then was rejuvenated and frozen. The cells were thawed, washed, and stored at 4 degrees C with CPDA-1 for an additional 14 days. The 24-hour viability of these cells was determined to be 74.0 +/- 5.1 percent. These findings show that the postthaw storage time of red cells can be increased greatly over the now-accepted 24 hours, if bacterial sterility can be assured.

Experimental Histomoniasis in Gnotobiotic Turkeys. I. Infection and Histopathology of the Bacteria-Free Host

Experimental Histomoniasis in Gnotobiotic Turkeys. I. Infection and Histopathology of the Bacteria-Free Host

Open healing of tuberculous cavities

Summary Fourteen patients treated with antibacterial drugs for pulmonary tuberculosis who had been sputum negative for periods of four to fourteen months and yet had persisting ring shadows at the site of radiologically proved cavities, were studied. The regimes of chemotherapy varied, but only 2 had had less than six months of continuous chemotherapy immediately before operation. Bronchograms, including broncho-tomograms were taken in all cases. Radio-opaque material outlined 13 cavities in 12 patients but failed to enter cavities in the remaining 2. In assessing the combined bacteriological and pathological results of 10 resected specimens, Melzer's histological criteria for complete healing are adopted but with the additional requirement of bacterial sterility. Only one cavity studied from a patient who had received eight months' chemotherapy was considered healed by these standards. In all the other specimens, most of them from patients treated for longer periods, there was some evidence of activity. The most striking feature was the varying degree of activity and healing seen in different parts of any one cavity. It is concluded that a pathological diagnosis of ‘healed cavity’ cannot be made with certainty unless detailed histological and bacteriological studies are combined.

Techniques for Rickettsial and Virus Cultivation

The following note describes modifications made in the Weigl method for cultivating rickettsiae and other small agents of disease within susceptible lice. Made primarily to facilitate isolations from diseased persons, they involve feeding larvae reared under sterile conditions on potentially infected blood. The technique is essentially as follows. From 200-400 adult lice (Pediculus vesti-menti) are washed for 2 minutes in 60% alcohol and subsequently with sterile physiological saline. They are then enclosed in a cage (6 × 3 × 1 cm) covered on one side with a fine net and containing within it the piece of cloth recommended by Weigl. The caged lice are fed twice daily on a healthy nourisher whose skin is first sterilized with 70% alcohol. Between feedings the cages are kept in sterile paper boxes at 34°C. Under these circumstances, masses of eggs are laid on the cloth. These eggs are removed and washed first with 30% alcohol or other suitable weak disinfectant and then with sterile saline, bacterial sterility of the washed eggs being checked by culturing the last drops of the saline wash on nutrient agar. The: sterile eggs are dried on sterile paper and incubated at 34°C in sterile net-covered cages, 200-400 eggs per cage. The eggs hatch after 4 to 6 days and the larvae are then ready for use. When used to isolate rickettsiae or other agents of disease from a patient, such a cage of larvae is fed sterilely and twice daily over a period of four to twelve days, depending on the disease being investigated. To do this, the net side of the cage is attached to the sterilized skin on the medial side of the forearm or shank. Sterilization is affected with ether followed by 70% alcohol, care being taken to be sure that the alcohol has completely evaporated before feeding begins. Bacterial sterility is routinely checked by seeding larval excrement to agar plates.

Chemical Treatment of Tumors. VI. Method Employed in Determining the Potency of Hemorrhage-Producing Bacterial Preparations

The method described in the previous paper (1) was found useful in discriminating between the activities of materials which differed widely in their potency in producing hemorrhage in mouse sarcomas. Difficulties were encountered, however, when the potencies were of the same order of magnitude. While at times the bioassays yielded good results, on other occasions discrepancies were noted, and repetition of determinations of potency sometimes gave widely discoidant results. Often a small dose produced hemorrhage in a greater percentage of the tumors than did large doses of the same material in the same assay experiment. Sometimes the tumors of an entire batch of mice appeared to be refractory to hemorrhage production. This paper reports a modified technique which yielded more satisfactory results. Testing of the tumor mash for bacterial sterility, increasing the number of mice used per dose level, examining the interior of the tumor for hemorrhage, and including a standard material for reference in each set of bioassays were the measures which were successful in reducing the discrepancies encountered previously.