Microbial Cell Concentration Research Articles

Gut microbiota response to in vitro transit time variation is mediated by microbial growth rates, nutrient use efficiency and adaptation to in vivo transit time

BackgroundTransit time is an important modulator of the human gut microbiome. The inability to modify transit time as the sole variable hampers mechanistic in vivo microbiome research. We singled out gut transit time in an unprecedented in vitro approach by subjecting faecal microbial communities from six individuals with either short, medium or long in vivo transit times, to three different colonic transit times of 21, 32 and 63 h in the validated human gut in vitro model, SHIME.ResultsTransit time was identified as the single most important driver of microbial cell concentrations (52%), metabolic activity (45%) and quantitative (24%) and proportional (22%) community composition. Deceleration of transit was characterised by a significant decrease of specific Bifidobacterium and Veillonella spp. and increase of specific fibre degrading bacteria and nutrient specialists, such as Bacteroides, Prevotella, Ruminococcus, Bilophila and Akkermansia spp. These microbial communities reached a higher population density and net carbohydrate fermentation, leading to an increased SCFA production at longer transit times. In contrast, the carbohydrate-to-biomass production efficiency was increased at shorter transits, particularly in well-adapted faecal microbiomes from donors with short in vivo transit. Said adaptation was also reflected in the carbohydrate-to-SCFA conversion efficiency which varied with donor, but also colon region and SCFA chain length. A long transit time promoted propionate production, whereas butyrate production and butyrate producers were selectively enriched in the proximal colon at medium transit time.ConclusionMicrobial growth rates and nutrient utilisation efficiency mediate the species-specific gut microbiota response to in vitro transit time variation, which is the main driver of in vitro microbial load, metabolism and community composition. Given the in vivo transit time variation within and between individuals, the personalisation of in vitro transit time based on in vivo data is required to accurately study intra- and inter-individual differences in gut microbiome structure, functionality and interactions with host and environmental modulators.1MQXL3e8MgVLC8CZ9wex6EVideo

Etiological features of urinary tract infections in pregnant women: current state of the problem

Background. Urinary tract infections in pregnant wo­men occupy a leading place in the structure of extragenital patho­logy. The purpose of the study is to determine the etiological features of urinary tract infections in pregnant women. Materials and ­methods. The study included 89 pregnant women with urinary tract infections. The material for microbiological research was urine, the samples of which were collected on the day of hospitalization of a pregnant woman before treatment. Identification of microorganisms was carried out with the use of ­MIKROLATEST® test systems (Erba Lachema, Czech Republic). Preparation of bacterial suspensions with a certain concentration of microbial cells was carried out using Densi-La-Meter electronic device (PLIVA-Lachema a.s., Czech Republic) according to the McFarland scale. Optical density was measured using a Multiskan EX microplate reader (type 355). Results. Escherichia coli, Proteus mirabilis, Proteus vulgaris, Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa are the leading pathogens of urinary tract infections in pregnant women of the Kharkiv region; Escherichia coli, Proteus mirabilis, Streptococcus pyogenes are predominant ones. The selected spectrum of etiological factors of urinary tract infections does not depend on the age of a pregnant woman. In the etiological structure of the urinary tract infections in pregnant women, Escherichia coli and Proteus mirabilis take the first and the second place during the first three trimesters of pregnancy. The third place in the first trimester belongs to Enterococcus faecalis, in the second trimester — to Proteus vulgaris and Streptococcus pyogenes, in the third trimester — to Streptococcus pyogenes. The etiological structure of urinary tract infections in pregnant women is determined by the topography of the inflammatory process. Thus, in women with asymptomatic bacteriuria, Proteus mirabilis takes the first place, Escherichia coli — the second place, Proteus vulgaris and Enterococcus faecalis — the third place; in acute pyelonephritis, the first place belongs to Escherichia coli, the second place to Streptococcus pyogenes, the third place to Proteus mirabilis, Enterococcus faecalis, Klebsiella pneumoniae; in urethritis, Escherichia coli takes the first place, Enterococcus faecalis, Klebsiella pneumoniae — the second place, Proteus mirabilis, Proteus vulgaris — the third place; in cystitis, the first place belongs to Escherichia coli, the second place to Proteus mirabilis, Proteus vulgaris, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa. Conclusions. The etiological features of urinary tract infections with different topography of the inflammatory process of bacterial etiology determined by the authors must be taken into account in the treatment of this category of pregnant women.

Expressing variable mass transfer coefficients for gas fermentation in trickle bed reactor

Gasification of lignocellulosic biomass and syngas biomethanation are promising technologies for producing biomethane. Trickle bed reactors (TBR) are efficient for syngas fermentation allowing for higher concentration of microbial cells and high surface area for mass transfer. Optimized scaling up and dimensioning of the reactor is crucial when it comes to industrial applications. The first step towards this goal and the scope of the present study is to develop a model able to simulate the mass transfer of gases to the liquid phase as a function of the operating conditions and reactor geometry. Experiments were performed in a lab scale TBR with co-current flow of syngas and water, representing the growth medium, to determine the volumetric mass transfer coefficient (kLa) of each gas compound and its dependence on the flowrate of syngas and water and reactor structural characteristics. The kLa achieved were in the range of 6.18–13.6 hr−1 for H2, 2.6–5.79 hr−1 for CO and 1.53–3.43 hr−1 for CO2. A dimensionless equation was fitted to the experimental data and a model was developed to predict kLa of a specific gas at various gas and liquid flowrates, and reactor configurations. A kinetic model was also developed to simulate the concentrations of syngas in gas phase and liquid phase by applying the kLa values predicted by the dimensionless correlation. Finally, the model was validated using experimental data obtained by mass transfer coefficient determination in pilot scale TBR with pure argon gas and literature data where the predicted kLa had a coefficient of variance of ± 15% and ± 25% respectively within a realistic range of liquid and gas flowrates.

A critical review on the effectiveness of microbial concentrations for enhancing self-healing in cement concrete and mortar

A significant consideration in the design of any structure is the choice of materials. Construction relies heavily on concrete due to its exceptional characteristics and is the primary material used to construct infrastructure across the globe. Moreover, these materials caused micro-cracks under cyclic loading due to tension, significantly reducing their life expectancy. Nowadays, structures have a decreased service life because of the degradation of their durability, strength, and many other attributes. In concrete or mortar structures, cracks are a significant concern as they cause deterioration and allow unwelcome chemicals to enter the system. Sealants and surface treatments have become increasingly popular in enhancing structural appearance in recent years. Despite their limitations, these products are unstable, costly, and contribute to higher pollution levels. When micro-cracks extend and end up inside reinforcement, not only will the concrete be destroyed, but its support will also be corroded by oxygen, water, and harmful gases. Access to the cracked zones may be difficult aside from these operations requiring capital and labour. Crack healing in concrete may be of assistance in reducing the growth and propagation of cracks in concrete. The application of such a concept has led to the development of concrete known as Bacterial Concrete, in which bacteria play the role of carrier and perform bio mineralization by decomposing urea and calcium to produce calcium carbonate (CaCO3), which is capable of filling cracks. Bacteria-induced carbonate precipitation is an environmentally friendly mechanism for autonomously sealing cracks. Many researchers around the world have recently been exploring this mechanism in depth. This paper aims to review the assessment of strength and other properties both in concrete and mortar in relation to specific microbial cell concentrations and to analyse its effectiveness in promoting crack healing and microstructural properties.

Enhance the Growth of <i>Clostridium ljungdahlii</i> Microbial Cells by Modifying the Medium Composition and Trace Metals

Syngas fermentation is an alternative route that combines the advantages of thermochemical and biochemical processes have been proposed for biomass conversion to ethanol. One of the main obstacles to syngas fermentation is the low yield of ethanol, caused by the limited utilization of the syngas substrate due to low microbial cell concentration in the fermentation system. This research examined the modification of fermentation medium to improve microbial cell growth. The modifications were to increase the concentration of micronutrients/trace metals and macronutrients in the medium. The results showed that the maximum mass cell and maximum growth rate produced by microbial growth in the modified trace metal medium were 0.63 g/L and 0.0076 h-1, while in a modified macronutrient medium were 0.97 g/L and 0.0298 h-1. Modification of the macronutrient medium was able to increase the yield of biomass, but the opposite occurred in the modification of the trace metals. Meanwhile, the maximum concentration of ethanol from syngas fermentation in the modified macronutrient medium was lower than the concentration of ethanol in the standard medium.

Bioconversion of Bottom Sediments Using Mesophilic Prokaryotes of the Genus Bacillus spp.

The aim of the study was to develop a scientific and technical justification for the bioconversion of bottom sediments using mesophilic prokaryotes. The main component of the bottom sediment bioconversion technology includes microorganisms of the genus Bacillus spp., isolated in a pure culture from the microbial community of bottom sediments. Bacilli are highly resistant to natural (climatic) conditions, anthropogenic factors, elevated concentrations of pollutants and are able to adapt to their effects. As a result of the laboratory experiment, work was carried out on the selection of factors that ensure the intensity of microbial bioconversion of bottom sediments. In order to achieve the maximum effect from the enzymatic and biochemical activity of bacilli, kinetic characteristics associated with the achievement of maximum values of optical density, microbial cell concentration and growth rate were studied. After analyzing a number of the above indicators, it is concluded that the method of processing bottom sediments using mesophilic prokaryotes of the genus Bacillus spp. will allow in the future to obtain an environmentally friendly and mineral-saturated product for further use in the practice of crop production.

Ways to reduce the labor intensiveness of applying the method of optical estimation of microbial cell concentration in suspension.

The labor intensity (in hours) of the optical method of microbial cell counting in suspension compared to the method of microbial cell counting using a Goryaev chamber is evaluated. The relevance of assessing the production labor intensity of microbial cell counting methods in suspension is related to the need to use them in many studies. Often the commonly used methods are too labour-intensive, time-consuming, or require expensive equipment. A comparative experiment was carried out with our previously developed "Method for optical estimation of microbial cell concentration in suspension" (Priority certificate No. 2016141859 dated 25.10.2016) and the method of microbial cell counting using a Goryaev chamber. Production labor intensity of the measurements performed was calculated in hours according to the formula: Tp=Tt+Tob, where Tp is production labour input, Tt is technological labour input, Tob is maintenance labour input. Technological labour input of measurements with use of Goryaev's chamber made up 32,18 ± 0,95, whereas with optical method - 1,03±0,06 (reliability of differences at p<0,01) at amount of measurements n = 100. Labour input of service at optical method 0,24 ± 0,03, at application of method with use of Goryaev chamber 0,15±0,01 hours. Labour input of measurements of concentration of microbial cells in suspension at application of method of measurement with Goryaev chamber remains (p<0,01) higher than at an optical method of estimation, 32,33±0,96 and 1,27±0,05 hours accordingly. When using the optical method of concentration estimation in the suspension it is necessary to carry out not a small amount of necessary mathematical calculations, which in the future, probably, corrected by creating a special program for a personal computer. The labour input of results obtained by measuring by optical evaluation of the concentration of microbial cells in suspension is lower than that obtained by using a measurement method using a Goryaev chamber. Taking into consideration that its implementation does not require purchase of special equipment as in turbidimetry, its cost-effectiveness compared to existing ones is obvious.

Microbial Water Quality through a Full-Scale Advanced Wastewater Treatment Demonstration Facility.

The fates of viruses, bacteria, and antibiotic resistance genes during advanced wastewater treatment are important to assess for implementation of potable reuse systems. Here, a full-scale advanced wastewater treatment demonstration facility (ozone, biological activated carbon filtration, micro/ultrafiltration, reverse osmosis, and advanced oxidation) was sampled over three months. Atypically, no disinfectant residual was applied before the microfiltration step. Microbial cell concentrations and viability were assessed via flow cytometry and adenosine triphosphate (ATP). Concentrations of bacteria (16S rRNA gene), viruses (human adenovirus and JC polyomavirus), and antibiotic resistance genes (sul1 and bla TEM ) were assessed via quantitative PCR following the concentration of large sample volumes by dead-end ultrafiltration. In all membrane filtration permeates, microbial concentrations were higher than previously reported for chloraminated membranes, and log10 reduction values were lower than expected. Concentrations of 16S rRNA and sul1 genes were reduced by treatment but remained quantifiable in reverse osmosis permeate. It is unclear whether sul1 in the RO permeate was from the passage of resistance genes or new growth of microorganisms, but the concentrations were on the low end of those reported for conventional drinking water distribution systems. Adenovirus, JC polyomavirus, and bla TEM genes were reduced below the limit of detection (∼10-2 gene copies per mL) by microfiltration. The results provide insights into how treatment train design and operation choices affect microbial water quality as well as the use of flow cytometry and ATP for online monitoring and process control.

Оценка экспрессии гена ctxA штамма Vibrio cholerae 569В при производстве холерной вакцины

Cultivation of production strains of Vibrio cholerae is one of the most important stages in the production of cholera bivalent chemical vaccines. In the production of immunobiological preparations, it is necessary to use producer strains with stable properties that persist in a number of generations. The aim of this study was to evaluate the expression of the ctxA gene using molecular genetic methods of the V. cholerae 569B producing strain. By PCR with electrophoretic accounting of the results, the presence of this strain of the ctxA gene in the chromosome was established. The expression of this gene in all hourly samples of culture fluid was recorded by the method of PCR with reverse transcription, taking into account the results in real time and digital drip PCR. Immunochemical methods confirmed the presence of cholera toxin in the samples, with a maximum mark at the ninth hour, which corresponded to the maximum indicators of the “concentration of microbial cells”. Thus, molecular genetic methods have shown the stability of the production strain V. cholerae 569B in samples of 4 independent cultivations.

Моделирование хронического остеомиелита в эксперименте

Introduction. In the existing models of osteomyelitis, there is no unified scheme for creating a pathological focus. To obtain reliable comparative data from Introduction There is no unified scheme for creating a pathological site in the existing osteomyelitis models. The location, the size of the defect, the dosage of the infecting agent and the carrier material are to be standardized to facilitate reliable comparative data from different authors and reproducibility of the experimental model. The objective was to demonstrate experimental results of simulated chronic osteomyelitis using a unified scheme for creating a pathological site. Material and methods An identical defect was simulated in the proximal tibia metaepiphysis of 15 rabbits using a four-sided cone-shaped drill with a diameter of 0.5 cm and a limiter to a depth of 0.5 cm. An allobone fragment impregnated with Staphylococcus aureus suspension with a microbial cell concentration of 1.0 × 108 CFU/mL was placed into the defect site. A part of sutures was removed from the middle third of the wound and the edges were diluted to initiate a fistula course at 3 postoperative days. An experimental model of chronic osteomyelitis was developed using unified parameters of location, defect size, dosage of the infecting agent and carrier material. The method was technically simple, required no additional infection and provided a chronic osteomyelitic process. Observation period was 21 days. The control of the model formation was produced through clinical observation, inflammatory changes in the peripheral blood, bacteriological, radiological and pathomorphological examinations. Results Postoperatively, the animals demonstrated a decreased physical activity, increased body temperature, impaired function of the operated limb, a non-healing fistula with an abundant purulent discharge of curd consistency formed at the site of the postoperative wound. Computed tomography showed a cavity with irregular sclerotic edges filled with multiple bone sequesters, edema of adjacent soft tissues and fistula at 21 postoperative days. Leukocytosis was observed in the peripheral blood. Bacteriological examination of the wound discharge showed growth of Staphylococcus aureus. Pathomorphological investigation indicated chronic osteomyelitis with bone defects in the proximal metaepiphysis of the tibia and necrotic areas, pronounced leukocyte infiltration, fragments of dissolving bone tissue, growth of connective tissue surrounding foci of chronic purulent inflammation. An experimental model of chronic osteomyelitis was developed using unified location parameters, defect size, dosing of the infecting agent and carrier material. The method was technically simple, required no additional infection and facilitated formation of a chronic osteomyelitic process for 21 days. Discussion We used allobone in our model to cause infection by impregnation of microbial suspension without additional removal of the carrier. The amount of infecting suspension to initiate osteomyelitic process to be absorbed by the allobone and avoid the death of the animal from septic complications was determined in the course of the study. For passive drainage of the wound, a fistula course was provided and its functioning maintained, with the osteomyelitic focus localized and survival of animals ensured throughout the experiment. Conclusions An experimental model of chronic osteomyelitis was demonstrated using a unified scheme for a pathological focus. The model allowed us to avoid generalization of the osteomyelitic process, ensure the survival of animals throughout the experiment and simulate the process being consistent with pathomorphological changes characteristic of human chronic osteomyelitis.

The synthesis, antimicrobial activity and docking studies of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin- 4(3H)-ones with acetamide and 1,2,4-oxadiazol-5-ylmethyl substituents

Aim. To synthesize, study the antimicrobial activity and suggest antimicrobial activity mechanism for the novel derivatives of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4(3H)-one. Results and discussion. As the result of the targeted modification of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]-pyrimidin-4(3H)-one in position 3 with acetamide and 1,2,4-oxadiazol-5-ylmethyl substituents, the compounds, which demonstrated better antimicrobial activity in the agar well diffusion assay than the reference drug Streptomycin, were obtained. To elucidate the mechanism of action of the novel compounds, the docking studies were con-ducted to the active site of the 16S subunit of ribosomal RNA, the proven target for aminoglycoside antibiotics, as well as tRNA (Guanine37-N1)-methyltransferase (TrmD), which inhibitors were considered as a new potential class of antibiotics. Experimental part. By the interaction of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4(3H)-one with a series of N-arylchloroacetamides and 3-aryl-5-(chloromethyl)-1,2,4-oxadiazoles in DMF in the presence of K2CO3 the target compounds were obtained. The antimicrobial activity was assessed by the agar well diffusion method. The concentration of microbial cells was determined by the McFarland standard; the value was 107 cells in 1 mL of the media. The 18 – 24 hour culture of microorganisms was used for tests. For the bacteria cultivation, Müller-Hinton agar was used, Sabouraud agar was applied for C. albicans cultivation. The compounds were tested as the DMSO solution with the concentration of 100 µg/mL; the volume of the solution was 0.3 mL, the same volume was used for Streptomycin (the concentration 30 µg/mL). The docking studies were performed using Autodock Vina. Crystallographic data for the complexes of Streptomycin with the 16S subunit of ribosomal RNA (1NTB) and its active site, as well as for tRNA (Guanine37-N1)-methyltransferase (EC 2.1.1.228; TrmD) (5ZHN) and its active site were obtained from the Protein Data Bank.Conclusions. It has been determined that 2-[6-(1H-benzimidazol-2-yl)-5-methyl-4-oxothieno[2,3-d]pyrimidin-3(4H)-yl]-N-[4-(ethoxy)phenyl]acetamide, which is the most active as an antimicrobial agent among the compounds tested, also shows the best binding activity towards the active site of tRNA (guanine37-N1)-methyltransferase.

Bactericidal efficiency of preparation based on essential oils used in aerosol disinfection in the presence of poultry

A disinfectant was created for aerosol disinfection of premises in the presence of poultry, which will help reduce microbial contamination of premises, increase survival, weight of poultry and economic efficiency of meat production in general. The preparation based on essential oils can be used for disinfection in the presence of poultry and at the same time exhibits a therapeutic and prophylactic effect on respiratory infections. This disinfectant has a colloidal solution of silver (Ag), benzalkonium chloride and essential oils of thyme, fir and eucalyptus. The preparation based on essential oils contains (per 100 g): benzalkonium chloride – 16.0 g; thyme oil – up to 2.0 g; eucalyptus oil – up to 2.0 g; fir oil – up to 2.0 g; colloidal solution of silver (Ag) – 20–30 mg; distilled water - up to 100 cm3. Aerosol sanitation of indoor air was carried out with 0.3% solution of preparation in the period before housing poultry and once a day from the 20th to the 35th day of growing broilers with aerosol cold mist generator Dyna-Fog Tornado (model 2897, construction type – ULV-electric spray gnerator, manufacturer – Curtis Dyna-Fog, Ltd., USA) at a dose of 50.0 cm3 per 1 m3 at an exposure of 60 minutes. The size of the aerosol particles is 20 μm. On days 1, 4, 8, 11, 15, 28, 37, and 42, the chickens were weighed, and the blood was taken for examination. Blood was examined to study the number of red blood cells, hemoglobin content, the bactericidal activity of blood serum, phagocyte activity of leukocytes, lysozyme activity of blood serum. According to the results of the research, the technological modes of air disinfection of poultry premises in the presence of broiler chickens were substantiated during the use of preparation, which contains nanoparticles (NP) of silver, benzalkonium chloride and essential oils. The optimal mode of aerosol treatment of poultry houses using a 0.3% solution preparation based on essential oils is 50 mL/m3 of a room with a 60-minute exposure. The use of air disinfection in the presence of chickens during broiler rearing and one treatment per day from 20 to 35 days of the chickens’ growth reduced the microbial pollution of indoor air. Thus, the concentration of microbial cells in the room where the chickens were kept was 230.2 ± 15.6 thousand microbial cell/m3. Sixty minutes after disinfection, the concentration decreased to 1.4 ± 0.4 thousand microbial cell/m3. In addition, the bodyweight of chickens at 6 weeks increased by 449.4 ± 16.3 g (15.9%) compared with the controls. The method and mode of air treatment did not adversely affect the development of the internal organs of the poultry and their physiological state, which is confirmed by studies of the morphological parameters of the chicken blood. The data obtained indicate a positive effect of the developed methods and modes of aerosol air treatment with the preparation based on essential oils on the growth and development of broilers.

Assessment of Biolog EcoplateTM method for functional metabolic diversity of aerotolerant pig fecal microbiota

In the last decades, gut microbiota and its role in mammal host development and health have been increasingly investigated. Metabolites produced by gut microbiota can affect intestinal homeostasis and immune system maturity and activation, and in turn, they can influence the health and growth performance of livestock. Therefore, a better understanding of the functional metabolic capability of the gut microbiota would be appreciated by the scientific community. In this study, the BiologTM Ecoplates technology was applied for studying the metabolic potential of the aerotolerant microbial community of pig fecal samples, evaluating the interference of different storage conditions and cell concentrations. The length of time for which a fecal sample maintained detectable and unchanged microbial metabolic activity was also investigated. Two assays aimed to evaluate differences in the metabolic activities between fresh and snap-frozen fecal samples at different dilutions and at different lengths of times of preservation at −80°C were carried out. The biodiversity and the predicted functionality of the entire bacterial community through a targeted metagenomic approach were also explored. The results highlighted that snap freezing of fecal samples preserved the metabolic activity of the microbial community when compared to fresh feces. Sample storage at −80 °C did not significantly affect the metabolic activity of the microbial community, which was stable for 150 days. Furthermore, the highest metabolic activity was detected with 1:2 to 1:5 dilutions of the stock suspension. BiologTM Ecoplates technology is a rapid and useful method to explore microbial communities’ metabolism in animal fecal samples contributing to investigate host animal physiology.Key points• Freezing of samples can preserve the functional activity of the aerotolerant microbial community for 150 days.• The concentration of microbial cells strongly influences metabolic activity detection.• Sequencing coupled with the BiologTMEcoplates could be a strategy to evaluate the metabolic potential of the microbiota of the fecal sample.Graphical abstract

A STUDY OF THE EFFICIENCY OF CULTURE MEDIA FOR THE RECOVERY OF LYOPHILIZED PATHOGENIC BACTERIA

Today, the most appropriate way of long-term storage of most microorganism strains in collections is their lyophilization. The rate and quality of the recovery of biological properties in lyophilized bacterial cultures depend on the quality of culture media used for this purpose. The purpose of this study was to investigate the effectiveness of culture media for the recovery of lyophilized pathogenic bacteria. The strains of Salmonella typhimurium, Pasteurella multocida, Yersinia pseudotuberculosis, and Staphylococcus aureus from the collection of the Ukrainian Laboratory of Quality and Safety of Agricultural Products of the National University of Life and Environmental Sciences of Ukraine were used in the study. In order to restore lyophilized bacterial cultures, several variants of culture media were used: meat-peptone broth (Pharmactive, Ukraine) with the addition of 5% equine serum and 0.5% glucose; meat-peptone broth (Pharmactive, Ukraine) with the addition of 5% bovine serum and 0.5% glucose; meat-peptone broth (Pharmactive, Ukraine) with the addition of 5% bovine serum and 0.5% glucose; brain-heart infusion broth (HiMedia, India) and tryptone soya broth (HiMedia, India). Faibich protective medium was used as a cryoprotectant. It was found that in the process of sublimation and cryopreservation of cultures of Salmonella typhimurium, Pasteurella multocida, Yersinia pseudotuberculosis, and Staphylococcus aureus, the loss of viable microbial cells was 99.91–99.987%. The concentration of viable microbial cells in lyophilized cultures of Salmonella typhimurium, Pasteurella multocida, Yersinia pseudotuberculosis, Staphylococcus aureus was recorded in the range of 1.4 × 10 4 –1.7 × 10 6 CFU/cm 3 . During a single passage on different culture media, obtained biomass of the studied cultures was in the initial titer x × 10 9 . The highest productivity in recovered cultures was obtained using brain-heart infusion broth manufactured by HiMedia, India (concentration of live microbial cells in experimental cultures was recorded in the range of 5.7–6.4 × 10 9 CFU/cm 3 ). Keywords : Salmonella, Pasteurella, Yersinia, Staphylococcus, nutrient media, lyophilization

Improvement of Microbial Cell Concentration Technology in the Production of Live Plague Vaccine in the Form of Orodispersible Tablets

The aim of the study was to improve the procedure for the Yersinia pestis EV strain cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit.Materials and methods. The study used the vaccine Y. pestis EV strain derived from NIIEG cell line. Submerged cultivation of the native culture was performed using BIOR-0.25 reactor with automated control system. Microbial suspension concentrate was produced through microfiltration applying (Adaptive filtration system) AFS-009 and AFS-020 installations. The content of live microbial cells was determined by cytorefractometry. Assessment of the resistance of Y. pestis EV strain cells to technological factors was performed by photometric registration of changes in the optical density of bacterial suspension during the lytic response of cells to sodium dodecyl sulfate. Physical-chemical and immunobiological properties of the dry live plague vaccine were determined in accordance with the pharmacopoeial item.3.3.1.0021.15 of the State Pharmacopoeia of the Russian Federation, 14th edition.Results and discussion. The design features of the equipment introduced made it possible to carry out membrane filtration of microbial suspension, using BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by routine and improved methods was more than 150 billion microbial cells per ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of AFS-009 and AFS-020 filter units did not significantly affect the morphometric properties and resistance of microbial cultures to extreme (technological) factors. Based on the experimental data, the mass balance of the membrane filtration process has been determined. The optimized technology gave 0.13 liter yield of concentrate from 1 liter of native culture, and the process duration was reduced to 5 hours, the yield of the finished product in one production cycle was increased by 3 times. Thus, the process of concentrating Y. pestis EV strain cells during the production of the tablet form of live plague vaccine has been enhanced. A comparative study of the morphometric properties and resistance of plague microbe cultures to technological factors in the process of their concentration using optimized technology did not reveal any significant differences as compared to the routine one. Technological stage of concentrating has been reduced to 5 h term with a three-fold increase in the yield of finished product.

Dynamic Hydraulics in a Drinking Water Distribution System Influence Suspended Particles and Turbidity, But Not Microbiology

Spatial and short-term temporal changes in water quality as a result of water age and fluctuating hydraulic conditions were investigated in a drinking water distribution system. Online measurements of total and intracellular adenosine tri-phosphate (ATP), total and intact cell concentrations measured with flow cytometry (FCM), turbidity, and particle counts were performed over five weeks at five subsequent locations of the distribution system. The high number of parallel FCM and ATP measurements revealed the combined effect of water age and final disinfection on spatial changes in microbiology in the system. The results underlined that regular daily dynamics in flow velocities are normal and inevitable in drinking water distribution systems, and significantly impact particle counts and turbidity. However, hydraulic conditions had no detectable impact on the concentration of suspended microbial cells. A weak correlation between flow velocity and ATP concentrations suggests incidental resuspension of particle-bound bacteria, presumably caused by either biofilm detachment or resuspension from sediment when flow velocities increase. The highly dynamic hydraulic conditions highlight the value of online monitoring tools for the meaningful description of short-term dynamics (day-scale) in drinking water distribution systems.

Wildland fire as an atmospheric source of viable microbial aerosols and biological ice nucleating particles.

The environmental sources of microbial aerosols and processes by which they are emitted into the atmosphere are not well characterized. In this study we analyzed microbial cells and biological ice nucleating particles (INPs) in smoke emitted from eight prescribed wildland fires in North Florida. When compared to air sampled prior to ignition, samples of the air-smoke mixtures contained fivefold higher concentrations of microbial cells (6.7 ± 1.3 × 104 cells m-3) and biological INPs (2.4 ± 0.91 × 103 INPs m-3 active at temperatures ≥ -15 °C), and these data significantly positively correlated with PM10. Various bacteria could be cultured from the smoke samples, and the nearest neighbors of many of the isolates are plant epi- and endophytes, suggesting vegetation was a source. Controlled laboratory combustion experiments indicated that smoke emitted from dead vegetation contained significantly higher numbers of cells, INPs, and culturable bacteria relative to the green shrubs tested. Microbial viability of smoke aerosols based on formazan production and epifluorescent microscopy revealed no significant difference in the viable fraction (~80%) when compared to samples of ambient air. From these data, we estimate each fire aerosolized an average of 7 ± 4 × 109 cells and 2 ± 1 × 108 biological INPs per m2 burned and conclude that emissions from wildland fire are sources of viable microbial aerosols to the atmosphere.

Prospects for Improving Quality Evaluation of the Live Brucellosis Vaccine in Terms of Specific Activity

Prophylactic immunisation against brucellosis is part of the National Immunisation Schedule for Epidemic Settings. The immunisation is performed with a live vaccine—a lyophilized suspension of the Brucella abortus strain 19 BА in a stabilizing medium. The paper presents the results of quality evaluation of 9 batches of live brucellosis vaccine that were submitted to the Testing Centre for Evaluation of Medicinal Immunobiological Products’ Quality of the Federal State Budgetary Institution “Scientific Centre for Expert Evaluation of Medicinal Products” of the Ministry of Health of the Russian Federation for assessment of the product’s compliance with the established specifications. The paper also presents the results of evaluation of the passport information provided by the manufacturer for these batches. There is no doubt about the need for objective quality evaluation of brucellosis vaccines as well as about the significance of its improvement.The aim of study was to assess the prospects for improving quality evaluation of live brucellosis vaccines in terms of Specific activity (concentration of microbial cells, number of living microbial cells, number of cutaneous doses).Materials and methods: specific activity (concentration of microbial cells and number of living microbial cells) was determined by visual and microbiological methods using the industrial reference standard of brucellosis vaccine OSO 42-28-396-2018, batch 6 and the bacterial suspension of the Brucella abortus strain 19 BА acquired from the joint stock company Scientific and Production Association “Microgen” in 2016. The number of cutaneous doses in the brusellosis vaccine was determined by the calculation method. Statistical processing of the results was performed using Microsoft Excel.Results: there was a mismatch between the brucella concentration coefficient of 1.7×109 microbial cells/mL determined by comparison with the industrial reference standard of bacterial suspension turbidity, 10 IU and the actual concentration of microbial cells obtained in the study. According to preliminary results, the brucella concentration coefficient corresponding to the industrial reference standard of bacterial suspension turbidity, 10 IU can reach 3.0×109 microbial cells/mL.Conclusions: the obtained results can serve as a basis for amending the data on the brucella concentration coefficient in the Passport and the Instructions for use of the industrial reference standard of bacterial suspension turbidity, 10 IU, as well as the Specific activity section (concentration of microbial cells, number of living microbial cells, number of cutaneous doses) of the established specifications for the brucellosis vaccine. Before amending the information on the brucella concentration corresponding to 10 IU in the Passport and the Instructions for use of the reference standard of bacterial suspension turbidity (OSO 42-28-85P), additional studies should be performed with other types of brucella.

Research of the inactivation of pathogens in water under exposure to low temperature plasma

There was investigated the effect of barrier and spark discharge low temperature plasma on water containing the cells of Escherichia coli (Escherichia coli), hay bacillus (Bacillus subtilis) and yeast (Saccharomyces cerevisiae). There was shown a general decline in the concentration of viable microbial cells after the treatment of suspensions. There was especially marked the detrimental effect of the method on the viability of sanitary-indicative coliform bacteria in the water.

Feedback regulation mode of gene circuits directly affects the detection range and sensitivity of lead and mercury microbial biosensors

Whole cell biosensors offer high potential for the detection of heavy metals in a manner that is simple, rapid and low-cost. However, previous researchers have paid little attention to the impacts of construction models on the performance of these biosensors, thereby limiting the achievement of rational design and the optimization of detection characteristics. Herein, for the first time, three basic models of lead and mercury detection circuits, namely feedback coupled, uncoupled and semi-coupled models, have been constructed and compared to explore the effects of uncoupling the topology of sensing circuits on the reporter signals. The results demonstrated that the uncoupled model had better sensitivity for both lead (50 nM) and mercury (1 nM), while the feedback coupled circuits had a wider detection range for mercury (10 nM - 7.5 μM). Introducing the semi-coupled model into the comparison revealed that both the type and location of promoters for regulatory protein genes were key factors for sensitivity. Moreover, the detection characteristics of the uncoupled biosensors were robust, as conditions such as induction time, the concentration of microbial cells, and the concentration of antibiotics had little interference on the performance of the microbial biosensors. This study also established a novel and simple pre-treatment method for sample detection by biosensors. When the uncoupled microbial biosensor was put into practice, the concentration levels of mercury in milk and lead in sewage were determined quickly and accurately. Our study, therefore, provides a strategy for the rational design of whole cell heavy metal biosensors and has developed the potential of their application.