[Comparative Genotyping of Staphylococcus aureus Strains Isolated from Skin Lesions, Nasal Cavities, and Feces of Children with Atopic Dermatitis].

Background: The lesion of skin of the majority atopic dermatitis patients is chronically colonized by bacteria belonging to the species Staphylococcus aureus. Topical antibacterial and anti-inflammatory therapy treatment are often ineffective due to fast recolonization by S. aureus and exacerbation of allergic process. Aims: Our aim was to determine a frequency of S. aureus colonization in skin lesions, mucous membranes of the nasal cavity and intestine of children with atopic dermatitis, to compare the genotypes of Staphylococcus aureus strains isolated from different biotopes of atopic dermatitis patients, and to clarify whether the intestinal and nasal cavities microbiota may act as a source of S. aureus recolonization of skin lesions. Materials and Methods: Bacteriological examination of fecal samples, skin, and nasal swabs was conducted in 38 atopic dermatitis patients. The pure bacterial cultures of S. aureus were identified using API Staph (Biomerieux, France) and Vitek 2 MS (Biomerieux, France). Isolates of S. aureus were subjected to genotyping by analysis of rRNA internal 16S-23S rRNA spacer regions and high resolution melting analysis (HMR) of polymorphic spa X-regions. Results: 99% S. aureus strains were successfully identified using MALDI-TOF mass-spectrometry. S. aureus cultures were isolated from all biotopes in 31,6% of children, from skin and nasal cavities ­ in 42% of cases, from skin and feces ­ in 2,6% of cases, only from skin ­ in 10,5%, from nasal cavities and feces ­ in 2,6%, and only from nasal cavities ­ in 2,6% of cases. In 8% of children, S. aureus was not detected in any of the biotopes. Genotyping of the isolates enabled the detection of 17 different genotypes. A match between the genotypes of skin and nasal strains, and skin and fecal strains was observed in 88% and 61% of the cases respectively. Conclusions: The observed a high-frequency matching genotypes suggests the possibility of migration of S. aureus strains inside biotopes in humans and the absence of specialization to colonization of any of the niches.

[Species Diversity of Bifidobacteria in the Intestinal Microbiota Studied Using MALDI-TOF Mass-Spectrometry].

BACKGROUND: The members of genus Bifidobacterium represent a significant part of intestinal microbiota in adults and predominate in infants. Species repertoire of the intestinal bifidobacteria is known to be subjected to major changes with age; however, many details of this process are still to be elucidated. OBJECTIVE: Our aim was to study the diversity of intestinal bifidobacteria and changes of their qualitative and quantitative composition characteristics during the process of growing up using MALDI-TOF mass-spectrometric analysis ofpure bacterial cultures. METHODS: A cross-sectional study of bifidobacteria in the intestinal microbiota was performed in 93 healthy people of the ages from 1 month to 57 years. Strains were identified using Microflex LT MALDI-TOF MS, the confirmation was performed by 16S rRNA gene fragment sequencing. RESULTS: 93% of isolated bifidobacterial strains were successfully identified using MALDI-TOF mass-spectrometry. At least two of the strains from each species were additionally identified by 16S rRNA gene fragment sequencing, in all of the cases the results were the same. It was shown that the total concentration of bifidobacteria decreases with age (p<0.001) as well as the frequency of isolation of Bifidobacterium bifidum (p=0.020) and Bifidobacterium breve (p<0.001), and the frequency of isolation of Bifidobacterium adolescentis, increases (p<0.001), representing the continuous process of transformation of microbiota. CONCLUSION: The method of MALDI-TOF mass spectrometry demonstrated the ability to perform rapid and reliable identification of bifidobacteria that allowed the study of changes in the quantitative and qualitative characteristics of human microbiota in the process of growing up.

[Impact of herpesvirus infections on the level of proinflammatory cytokines in premature neonatal infants].

The aim of this study was to estimate the frequency of HSV and/or CMV among the pathogens causing intrauterine infections (IUI) and to investigate their impact on the level of proinflammatory cytokines in premature neonatal infants. Examinations were performed in 3 neonatal groups: 1) premature neonates with clinical manifestations of IUI; 2) those without IUI; 3) full-term newborns. In group 1, viral (HSV and/or CMV) and bacterial infections were detectable with the same frequency. Quantitative analysis of plasma IL-6 and IL-8 levels and the induced production of these cytokines by blood cells in vitro showed that in Group 1 neonates, IL-6 and IL-8 concentrations were substantially higher and the induced production of these cytokines was lowerthan those in Group 3. The detection of HSV and/or CMV markers in premature newborn infants was attended by a statistically significant rise in plasma IL-6 levels; the identification of the opportunistic bacterial microflora correlated with the higher concentration of IL-8. In Group 1, wiferon produced an immunomodulatory effect, by lowering IL-8 concentrations to the level observed in Group 3.

[Clinical-encephalographic evaluation of preterm children treated with cytoflavin during the first year of life].

An aim of the study was the clinical and neurophysiological evaluation of the effect of cytoflavin and reanimation measures on the rate of nervous and mental development of preterm newborns with cerebral ischemia, II--III stage, during the first year of life. One hundred and twenty children with gestational age 28--36 weeks and body mass 1060-3150 g were studied. In the main group (n=61), the complex treatment included cytoflavin in dosage 2 ml/kg/day, intravenously during 5 days. The control group (n=59) received basic treatment without cytoflavin. The follow-up till the age of 1 year comprised the global assessment of state and neurological status using quantitative measurements of muscle postural tone on the INFANIB scale, psychomotor development (BSID-II) and assessment of CNS functional state by analyzing computed EEG signals during physiological sleep. The results obtained demonstrated the fast normalization of all studied measures of development of children treated with cytoflavin compared to those of the control group.

[Controlled mechanical lung ventilation with guaranteed volume in newborns].

Mechanical ventilation with guaranteed volumes combines advantages of time-cyclic ventilation with limitation of pressure and constant flow, with volume control ventilation. This symbiosis allows to choose the optimal, stable breathing volume of the newborn, while maintaining pressure control. The review presents the principles of work, the main advantages and limitations of ventilation systems with the guaranteed volumes, as well as results and analysis of studies.

[Current approaches to cerebroprotective treatment of premature newborns in reanimation and intensive care departments].

This work was conducted in the frames of a multicenter clinical trial. The aim was to study efficacy of cytoflavin (infusion solution) in the prevention and treatment of posthypoxic CNS lesions in premature newborns. The study included 120 premature newborns (gestation period 28-36 weeks) who was born in severe distress and needed the intensive therapy after primary reanimation measures. Cytoflavin was prescribed in the first 2-4 h after the delivery to 61 newborns. The control group included 59 newborns who did not receive the drug. To assess treatment efficacy, the determination of some plasma neurospecific proteins (GFAP, NSE, MBP) was carried out along with standard clinical/instrumental and laboratory monitoring. The results revealed the marked cerebroprotective effect of cytoflavin. The significantly higher rate of normalization of KOC, pO2, PCO2 and elimination of lactate acidosis that led to the reduction of severity and frequency of ischemic and hemorrhagic CNS lesions as well as lower levels of plasma neurospecific proteins were seen in the main group compared to the control one.

Molecular genetic study of species and strain variability in bifidobacteria population in intestinal microflora of breast-fed infants and their mothers.

Qualitative and quantitative composition of enteric bifidoflora was studied in a group of 13 mother-infant pairs. Pure cultures of Bifidobacterium strains were isolated from feces and their species were identified by PCR with species-specific primers or by partial sequencing of 16S rDNA. The strains were compared by REP-PCR. The most incident Bifidobacterium species in mothers were B. longum and B. adolescentis. The infants were mainly colonized by B. bifidum and B. longum. The mother and her baby were colonized by the same Bifidobacterium species in 9 of 13 cases. In 5 (38.5%) of these cases, these pairs of strains were identical by their REP-PCR profiles. These strains belonged to B. longum in one case, B. bifidum in 3 cases, and B. adolescentis in 1 case. Our results support the hypothesis on early colonization of infants with maternal bifidobacterium strains.

Monitoring of motor disorders in 7-day-old rats with severe hypoxic-ischemic injury of the brain.

Seven-day old rats were subjected to unilateral ligation of the common carotid artery followed by 2-h exposure to a gas mixture consisting of 8% oxygen and 92% nitrogen. Locomotor function of rats was monitored weekly. Functional deficit in these animals persisted for at least 3 months. The exercise tests of rotarod, hanging, and narrowing track were most informative. Our results can be used in preclinical studies of new drugs for the therapy of perinatal brain injury.

Relationship between suppression of E6 and E7 virus oncogenes and expression of apoptosis and cell cycle genes in cervical cancer culture.

The effects of short interfering RNA suppressing the expression of E6 and E7 human papilloma virus (type 18) on the expression of apoptosis and cell cycle genes were studied in HeLa cells. Changes in the transcription profiles were evaluated using DNA microarray and real-time reverse-transcription PCR. Cell transfection with anti-E6 and anti-E7 short interfering RNA moderately reduced the expression of mRNA for CDC25C, GRB2, GTSE1, and PLK1 genes and induced expression of CDKN1A (p21(CIP)) gene mRNA. In addition, culture proliferation was inhibited and morphological changes characteristic of differentiation and cell aging developed.

[Problems and prospects of experimental modeling of hypoxy-ischemic lesions in the central nervous system].

Perinatal hypoxy-ischemic brain lesions are one of the main causes of mortality and dysfunction of the central nervous system in the neonatal period accounting for high disability rate among survivors. Numerous animal models were proposed to study this problem in ante-, intra-, and neonatal periods of ontogenesis. This paper is devoted to the analysis of the adequacy of these models. The processes of brain development in laboratory animals are considered along with etiopathogenetic factors that can be reproduced on the models of perinatal hypoxy-ischemic lesions in CNS. The available data on such models and their correspondence to known clinical syndromes are summarized. Current trends in the development of new models of hypoxy-ischemic brain lesions are discussed.

[Peculiarities of microbial colonization of the intestinal tract in newborns and pre-term infants in intensive care units].

The aim of the study was to compare the development of intestinal microflora in clinically healthy newborns, born by mothers with physiological pregnancy, and in small premature infants, who were treated in intensive care units (ICU) using various regimens of antibacterial therapy. The study revealed that the most frequent bacteria found in the intestinal tract of healthy infants at the and of neonatal period were bifidobacteria, enterobacteria, and coagulase-negative staphylococci and enterococci. Together with large quantity of autochtonous bacteria, the study revealed conditionally pathogenic microorganisms, such as klebsiella and coagulase-positive staphylococci at the end of neonatal period. The intestinal microflora of premature infants in ICU, treated with a combination of third generation cephalosporins and aminoglycosides from the first hours of life, was characterized by total absence of indigenous microflora, and prevalence of enterococci and staphylococci. The results show that the first stage of antibacterial therapy of preterm infants in ICU should be based upon the principles of selective decontamination.

[Detection of the markers of herpes simplex virus and cytomegalovirus in newborns and infants].

A total of 111 children suspected for herpesvirus infection were examined. In blood and urine samples the infectious activity of herpes simplex virus (HSV) and cytomegalovirus (CMV) was detected by the rapid culture method (RCM) and the presence of virus DNA--by the polymerase chain reaction (PCR). HSV and/or CMV were detected by two laboratory methods in 57 examined children (51%). Of these, in 18 children (16.2%) both HSV and CMV were detected. The coincidence of the results of the detection of HSV and CMV by these two methods was observed in 72.4% and 75.2% of cases respectively. The comparative analysis of the detection of anti-CMV IgG and IgM was made with the use of commercial test systems produced bythe following manufacturers: "Vector-Best" and "Bioservice" (Russia), "HUMAN" and "Boehringer" (Germany). The effective detection of both anti-CMV (IgG and IgM) was ensured by the test systems "Boehringer". The test system "Vector-Best" for anti-CMV IgG proved to be not inferior as regards sensitivity and specificity. The German test systems demonstrated the highest specificity in the detection of low-avid antibodies. The data obtained in this study indicate that the detection rate of HSV and CMV markers in newborns and infants suspected for herpesvirus infection was, on the average, 20 - 40%. Reliable diagnostics in newborns and infants is possible only in the presence of the combination of at least 2 serological tests (the determination of antivirus IgM and IgG avidity) and 2 methods for the detection of direct herpesvirus markers (PCR and RCM).

[Quantitative laboratory methods for the diagnosis of cytomegalovirus infection in premature infants].

31 prematures with signs of the cytomegalovirus infection (CMV) were examined. The blood and urine samples were tested for direct viral markers, i.e. for infectious CMV by the rapid culture method (RCM) and for viral DNA by quantitative PCR. Besides, the parameters of the specific immune response were studied in the babies. CMV was detected by RCM and/or PCR in 25 of the 31 examined babies during their 1st life week. The highest content of CMV within the investigated samples, i.e. 100 antigen-containing cells per 2.5 x 10(5) culture cells and above 2000 copies/ml of viral DNA was detected in 8 (32%) children. The quantity of viral DNA did not exceed 1000 copies/ml and one to three of stained cells was detected by PCR in 13 (42%) children. A study of anti-CMV in sera revealed high-titer of AT IgG in all 30 children. High avidity of anti-CMV-IgG was demonstrated to correlate with a low viral load and a low CMV infection activity in the newborns. According to the results, at least 3 laboratory diagnosis tools should be used in the diagnosis, they are PCR, RCM and determination of the anti-CMV avidity.

[Search of promising strains of bifidobacteria and lactobacillus for the development of new biopreparations]. / Poisk perspektivnykh shtammov bifidobakterii i laktobatsill dlia razrabotki novykh biopreparatov.

For the first time the species composition of bifidobacteria and lactobacilli in clinically healthy young children has been studied. As revealed in this study, the dominating species of bifidobacteria are B. longum, B. adolescentis and B. infantis, while the dominating species of lactobacilli are Lactobacillus acidophilus and L. rhamnosus. In 83 isolated cultures of bifidobacteria and 34 isolated species of lactobacilli the activity of acid formation and the antagonistic activity with respect to Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Clostridium perfringens have been tested. B. longum strain 58B, B. infantis strain 37B, L. rhamnosus strain 12L and L. acidophilus strain 27L, typical for children of this age group, having good antagonistic activity and pronounced acid-forming properties, have been selected. These strains hold good promise to be used as the basis for the development of a complex probiotic preparation for correcting intestinal microflora in young children.

Neurological deficit and disturbances in higher nervous activity during modeling of perinatal hypoxic-ischemic damage to the central nervous system in rat pups.

Seven-day-old Wistar rat pups were subjected to unilateral occlusion of the common cerebral artery and maintained in oxygen-low atmosphere. Neurological and behavioral changes were monitored for 12 weeks. The survival rate of treated animals was 90%. Body weight gain in these rats was lower than in the control. Neurological deficit was maximum 1 week after treatment and slightly regressed by the 12th week. Locomotor activity in treated rats was higher than in controls. Administration of ketamine in subanesthetic doses caused permanent ipsilateral rotational asymmetry in animals. Spatial disorientation and cognitive deficit in rats with hypoxic-ischemic damage to the central nervous system were revealed in passive avoidance, Y-maze, and rotarod tests. The total area of the hemisphere decreased, while the area of the lateral cerebral ventricle increased at the side of occlusion over the first 4-5 weeks of postnatal development. The size of the ipsilateral hemisphere remained low in adult animals.

Enzyme immunoassay of NSE and GFAP as the criterion of dynamic evaluation of the rat blood-brain barrier in perinatal hypoxic ischemic injury of the CNS.

Enzyme immunoassay of the serum neurospecific antigens (gliofibrillar acid protein and neurospecific enolase) was used for evaluation of the resistance of the blood-brain barrier in Wistar rats with perinatal hypoxia and ischemia of the CNS. Perinatal hypoxia and ischemia of the CNS was modeled by two methods: ligation of the common carotid artery in 7-day-old rats followed by 3.5-h hypoxic hypoxia or 15-min anoxic exposure of fetuses isolated via hysterectomy on day 21 of gestation. Enzyme immunoassay of serum gliofibrillar acid protein and neurospecific enolase in control an experimental rat pups was carried out once a week during 3 months. In controls serum levels of gliofibrillar acid protein and neurospecific enolase virtually did not change during postnatal development, while in animals with cerebral hypoxia and ischemia induced in fetuses by both methods serum concentration of neurospecific enolase sharply increased 1 week after the injury and increased on weeks 6 and 10. The content of gliofibrillar acid protein was maximum on week 1 and later considerably varied, the peaks of its concentrations observed on weeks 3 and 8 preceded the increase in neurospecific enolase activity in peripheral blood.