Dried nutrient media for the cultivation of pneumococcus and meningococcus

Dried synthetic nutrient medium for the cultivation of meningococci and the accumulation of their biomass has been developed. The kinetics of the culture growth, changes in pH and in the content of dissolved oxygen in the medium during prolonged controlled processes of the cultivation of meningococci in a bioreactor with the use of this medium have been studied. The stable physico-chemical properties and composition of the polysaccharide-protein complex isolated from the biomass of meningococci grown in the above-mentioned medium have made it possible to use it for the preparation of the samples of group B meningococcal vaccine. In addition, dried semi-synthetic nutrient medium for the accumulation of pneumococci without the necessity of introducing blood or serum into the medium has been developed. As regards its biological properties, this newly developed medium make it not inferior to meat media, containing blood or serum, and ensures good yield of biomass.     

Optimization of isolation and cultivation of bacterial endophytes through addition of plant extract to nutrient media

Many endophytes have beneficial effects on plants and can be exploited in biotechnological applications. Studies hypothesize that only 0.001–1% of all plant‐associated bacteria are cultivable. Moreover, even after successful isolations, many endophytic bacteria often show reduced regrowth capacity. This research aimed to optimize isolation processes and culturing these bacteria afterwards. We compared several minimal and complex media in a screening. Beside the media themselves, two gelling agents and adding plant extract to media were investigated to enhance the number and diversity of endophytes as well as the growth capacity when regrown after isolation. In this work, 869 medium delivered the highest numbers of cultivable bacteria, as well as the highest diversity. When comparing gelling agents, no differences were observed in the numbers of bacteria. Adding plant extract to the media lead to a slight increase in diversity. However, when adding plant extract to improve the regrowth capacity, sharp increases of viable bacteria occurred in both rich and minimal media.     

Structure and function of Hib pili from Haemophilus influenzae type b

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 A in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 A along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.     

Determination of the bacterial contamination of nutrient media for the biosynthesis of antibiotics

Contamination of separate components of nutrient media used in biosynthesis of antibiotics was tested. It was shown that corn steep liquor, corn and soybean meals were the most contaminated components. The results provided determination of the nutrient media contamination levels by the additive factor with respect to the percentage of the components. The safe period of 2-3 hours for the storage of the nutrient media was determined. After that period the media contamination levels markedly increased.     

Dynamics of growth of Haemophilus influenzae serotype B and synthesis of capsular polysaccharide in the process of cultivation in a synthetic nutrient medium

The dynamics of H. influenzae, serotype b, growth and synthesis of their capsular polysaccharide in the synthetic nutrient medium, proposed by Herriot for noncapsular strains, was studied using 6 strains. The growth rate of H. influenzae, serotype b, and the amount of capsular polysaccharide, synthesized in the above mentioned medium, practically were not different from those in heart-brain broth (Difco). The possibility of minimizing the composition of Herriot's medium without any adverse effect on the amount of synthesized capsular polysaccharide was shown. As the result of these studies, the expediency of the cultivation of H. influenzae, serotype b, in the synthetic medium, intended for obtaining the preparations of capsular polysaccharide, was proved.     

Mechanism of DNA degradation by the ATP-dependent DNase from Hemophilus influenzae Rd.

The rate of production of acid-soluble material during degradation of duplex DNA by Hemophilus influenzae ATP-dependent DNAse (Hind exonuclease V) has been shown to be directly dependent upon the Mg2+ concentration in the reaction mixture. At high concentrations of Mg2+ (5 to 20 mM), DNA degradation to acid-soluble products is rapid and the rate of ATP hydrolysis is slightly depressed. At low concentrations of Mg2+ (0.1 to 0.5 mM), the enzyme rapidly hydrolyzes ATP and converts up to 35% of linear duplex DNA to single-stranded material while degrading less than 0.2% of the DNA to acid-soluble products. We refer to this enzymatic production of single-stranded DNA as the "melting" activity. Under the conditions of our assay, the initial melting reaction is processive, lasting about 70s on phage T7 DNA. Using DNAs with several different lengths, we have established that the duration of the initial reaction is dependent upon DNA length, requiring approximately 1 s per 0.18 mum. The products of the initial reaction on phage T7 DNA are somewhat heterogeneous, consisting of short duplex fragments approximately 0.5 mum long, purely single-stranded products up to 7 mum long, and longer duplex fragments 3 to 11 mum in length, some of which have single-stranded tails. Nearly half of the single-stranded material remains linked to a duplex segment of DNA after the inital processive reaction. We propose that Hind exo V initiates attack at the DNA termini and then acts in a processive manner, migrating along the DNA molecule, converting some regions to single-stranded material by the combined action of the melting activity and limited phosphodiester cleavage, while leaving other regions double-stranded. At the completion of its processive movement through a single DNA molecule, it is released and then recycles onto either intact molecules or the partially degraded products, continuing in this manner until the DNA is finally reduced to oligonucleotides.     

Inclusion of geothermal water in composition of nutrient medium for mycobacteria cultivation

New effective nutrient medium for isolation and cultivation of mycobacteria was developed. Time of their growth on the developed medium decreased by 3 to 11 days compared with control media (Finn II, Levenstein-Jensen). Rate of mycobacteria isolation increased by 20 to 40% compared with control media.     

On the sensitivity of forward scattering patterns from bacterial colonies to media composition

Morphology of colonies is important for taxonomy and diagnostics in microbiology where the response to environmental factors is sensitive enough to support discrimination. In this research, we analyzed the forward scattering patterns of individual Escherichia coli K12 colonies when agar hardness and nutrition levels were varied from the control sample. As the agar concentration increased from 1.2% to 1.8%, the diameter of the forward scattering patterns also increased for the same experimental condition which reflects that the colony thickness at the apex is greater for increased agar concentrations. Regarding nutrition, increasing dextrose resulted in smaller mean colony diameters while the mean diameters of the colonies were proportional to the yeast extract concentration up to 0.5%. The result reveals that ±0.3% agar concentration from the control sample is sufficient to create variations in the scattering patterns. For nutrition –0.25% of yeast extract showed significant variations while +0.25% from control sample showed minimal variations. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Trace elements in raw materials and nutrient media for cultivation of antibiotic-producing organisms

The contents of microelements such as Fe, Cu, Zn and As in samples of raw materials including food grade ones used in preparation of nutrient media for cultivation of antibiotic-producing organism and water were determined spectrophotometrically with using complex forming organic reagents. An original procedure for the sample combustion in the assay was developed. The natural background levels of the microelements in the nutrient media used in manufacture of some antibiotics were estimated.     

An NMR spectroscopic identity test for the control of the capsular polysaccharide from Haemophilus influenzae type b.

We describe the use of Nuclear Magnetic Resonance (NMR) spectroscopy to control the identity of purified bulk capsular polysaccharide [called poly(ribosylribitolphosphate) or PRP] from Haemophilus influenzae type b (Hib), and derivatised forms, used in the production of Hib polysaccharide-protein conjugate vaccines. We describe the approaches we have developed to validate this test.     

Vaccination of children born from HIV-infected mothers against Haemophilus influenzae type b infection

Course of postvaccinal period after injection of vaccine against Haemophilus influenzae type b administered simultaneously with vaccines of Russian national immunization schedule was studied in children born from HIV-infected mothers. Good tolerability of the vaccine administered concomitantly with diphtheria-tetanus-whole cell pertussis and inactivated polio vaccines (Imovax Polio), which is comparable with tolerability in healthy children, was demonstrated. Prevaccination titers of antibodies and their dynamics during immunization process were described. Increase of levels of antibodies was detected both in the group of children with perinatal contact with HIV infection and in the group of HIV-infected children.