Methodology for Integral Study of Antagonistic Activity of Normal Nasal Microbiota to Select Potential Probiotics Efficient in Eradication of Staphylococcus aureus.

The aim of this study was the development of a methodology for the integral study of the antagonistic activity of normal human microbiota against Staphylococcus aureus to enable direct selection (without prior isolation of pure cultures) of potentially highly efficient probiotic preparations. The selection of bacterial representatives of normal human nasal microbiota capable of antagonizing S. aureus was carried out using two complimentary methods: replica-plating and deferred antagonism procedures. The material of the anterior nares from healthy human subjects was plated onto the surface of different nutrient media agar plates followed by incubation under appropriate conditions. The grown bacterial colonies were replica-plated to Petri dishes with nutrient agar overlayed with a thin layer of a soft agar which contained the culture of an indicator S. aureus strain. This agar also supported the growth of potential probiotic strains. The potential probiotic strains were selected by their ability to suppress the growth of S. aureus around their colonies. Most active strains-inhibitors may be used to develop probiotic preparations with targeted activity against S. aureus.

[The microbiocenosis of the palatal tonsils in the practically healthy people]. / Mikrobiotsenoz nebnykh mindalin u prakticheski zdorovykh lits.

The objective of the present study was the investigation of the quantitative and qualitative composition of the cultured microorganisms isolated from the proximal parts of the palatal tonsil lacunes of the practically healthy people. The data obtained made it possible to characterize the microbiocenosis of the palatal tonsil lacunes in the practically healthy subjects at the age from 18 to 30 years. A total of 153 strains were identified with the use of mass spectrometry; they represent six genera of two phylotypes that occur in the form of associations comprised of 4-5 microorganisms with the predominance of the species of the genus Streptococcus. The results of the study expand our knowledge about the oropharyngeal microbiota and create the prerequisites for the better uundderstanding of the contribution of the microbiotic communities to the development of pathology of the upper respiratory tracts.

Noncontiguous finished genome sequence of Megasphaera sp. ASD88, isolated from faeces of a child with autism spectrum disorder.

We report here a draft genome sequence of Megasphaera sp. ASD88, a strain from the intestinal microbiota of a child with autism spectrum disorder, representing a previously undescribed species of the genus Megasphaera. The assembled sequence consists of 88 scaffolds, and the total size is 2.59 Mb.

[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.

Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

Draft Genome Sequence of Lactobacillus fermentum NB-22.

We announce here a draft genome sequence of Lactobacillus fermentum NB-22, a strain isolated from human vaginal microbiota. The assembled sequence consists of 190 contigs, joined into 137 scaffolds, and the total size is 2.01 Mb.

[Quantitative and qualitative composition of axilla microbiota in practically healthy individuals].

AIM: Characteristics of quantitative and qualitative composition of cultured microorganisms isolated from axilla skin of practically healthy individuals. MATERIALS AND METHODS: 77 practically healthy individuals aged 18 to 40 years were examined. Species identification of microorganisms was carried out byculture-morphologic, tinctorial and biochemical properties using time-of-flight mass spectrometer and rpoB gene amplification with subsequent direct sequencing. RESULTS: Quantitative evaluation of microbial composition of axilla skin microbiota in most of the practically healthy individuals varied in the 4-5 lg CFU/ml interval, whereas seeding of skin by this microbiota at the level of 8 lg CFU/ml was not detected. 158 strains of 24 microorganism species were identified in this biotope. Most of these strains (68.9%) belonged to Corynebacterium genus, 21.6% of strains--to Staphylococcus genus, 7.6% of strains--to Micrococcus genus and 1.9% of strains--Candida albicans. 16 species of corynebacteria were isolated with predomination of C. tuberculostearicum (40.3%), C. amycolatum (18.4%) and C. ureicelerivorans (14.8%) strains. The microbial landscape in most of the examined individuals (77.9%) was presented by microorganism association. CONCLUSION: Quantitative and qualitative species composition of cultured microorganisms isolated from axilla skin biotope of practically healthy individuals was characterized for the first time.

Production of biologically active scFv and VHH antibody fragments in Bifidobacterium longum.

Bifidobacteria constitute a significant part of healthy intestinal microbiota in adults and infants and present a promising platform for construction of genetically modified probiotic agents for treatment of gastrointestinal disorders. In this study, three strains of Bifidobacterium longum were constructed that express and secrete biologically active single-chain antibodies against human TNF-α and Clostridium difficile exotoxin A. Anti-TNF-α scFv antibody D2E7 was produced at the level of 25 µg L(-1) in broth culture and was mostly retained in the cytoplasm, while VHH-type antibodies A20.1 and A26.8 against C. difficile exotoxin A were produced at the levels of 0.3-1 mg L(-1) and secreted very efficiently. The biological activity of both antibody types was demonstrated in the mammalian cell-based assays. Expression of A20.1 and A26.8 was also observed in vivo after intragastric administration of transformed B. longum strains to (C57/BL6 × DBA/2)F1 mice. The obtained B. longum strains may serve as prototypes for construction of novel probiotic medications against inflammatory bowel disease and C. difficile-associated disease.

Draft Genome Sequence of Coprobacter fastidiosus NSB1T.

Coprobacter fastidiosus is a Gram-negative obligate anaerobic bacterium belonging to the phylum Bacteroidetes. In this work, we report the draft genome sequence of C. fastidiosus strain NSB1(T) isolated from human infant feces.

Draft Genome Sequences of Two Pairs of Human Intestinal Bifidobacterium longum subsp. longum Strains, 44B and 1-6B and 35B and 2-2B, Consecutively Isolated from Two Children after a 5-Year Time Period.

We report the genome sequences of four isolates of a human gut symbiont, Bifidobacterium longum. Strains 44B and 35B were isolated from two 1-year-old infants, while 1-6B and 2-2B were isolated from the same children 5 years later. The sequences permit investigations of factors enabling long-term colonization of bifidobacteria.

Bifidobacterium longum modified recombinant HU protein as a vector for nonviral delivery of DNA to HEK293 human cell culture.

Creation of effective nontoxic highly specific systems for nonviral transportation of DNA is one of the priority problems in the development of genotherapeutic methods. Chimerical recombinant proteins consisting of Antp and Tat protein cell-penetrating domains and Bifidobacterium longum DNA-binding histone-like protein HU were obtained. The resultant recombinant proteins bind to plasmid DNA in vitro and provide intracellular delivery and expression of the reporter genetic construction with GFP gene in cultured HEK293 human cells.

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.

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.

Heterologous expression of secreted biologically active human interleukin-10 in Bifidobacterium breve.

Construction of Bifidobacterium breve capable of production of secreted biologically active human interleukin-10 (hIL-10) is described. ORF coding for full-length mature human interleukin-10 was cloned into a series of expression vectors. This resulted in generation of translational fusions between hIL-10 and signal peptides sequences derived from Bifidobacterium breve genes sec2, apuB and B. adolescentis gene amyB under the control of constitutively active bifidobacterial promoter. We have shown that fusion to amyB signal peptide resulted in highest expression level of hIL-10 at the mRNA and protein level. Secreted hIL-10 was highly unstable in bifidobacterial culture supernatants in standard growth conditions. However, incubation of stationary cultures in buffered tissue culture medium resulted in production of stable biologically active hIL-10, albeit in low amounts (1.9 ng/ml).

[Expression of green fluorescent protein (GFP) gene of Aequorea victoria [correction of Aequoria victoria] in Lactobacillus plantarum bacterium ].

Results of development of shuttle expressing plasmid vector Escherichia coli-Lactobacillus which allowed high level expression of heterologous genes in lactobacilli are represented. Vector pTRKH2 which is able to replicate in E. coli and in wide range of Gram-positive bacteria was used as the base. In order to provide high level of cloned gene expression constitutive-active synthetic promoter, site of initiation of translation, and terminator of transcription were introduced in the vector. Functional activity of this vector was confirmed using green fluorescent protein (GFP) gene from Aequoria victoria. Transformation of model strain by gfp gene-carrying plasmid resulted in appearance of typical fluorescent phenotype.

Production of human basic fibroblast growth factor (FGF-2) in Bifidobacterium breve using a series of novel expression/secretion vectors.

Four E. coli-Bifidobacterium shuttle vectors were constructed using Bifidobacterium plasmids, pB44 and pB80. The vectors carry two bifidobacterial promoters, a signal peptide-encoding sequence, sec2, of Bifidobacterium breve, and a transcriptional terminator from hup gene of Bifidobacterium longum. Functionality of the constructs were tested using human FGF-2 gene. The expression of FGF-2 was detected by Western blotting in B. breve transformed with three of the vectors. The highest amount of FGF-2 was produced upon transformation with pESH86, which is a pB80-based plasmid carrying FGF-2 under control of a hup promoter (Phup). Similarly, the level of FGF-2 mRNA transcribed from pESH86 was approximately threefold higher, 882 +/- 70 AU (arbitrary units), when compared to those transcribed from pB44-based pESH46 (Phup) (289 +/- 65 AU) and pESH47 (Pgap) (282 +/- 37 AU). These results suggest the vectors have the potential for production of exported fusion proteins in bifidobacteria and the expression levels can be regulated through the employment of different bifidobacterial promoters and/or replicons.

[Bifidobacterial plasmids and their application to genetic engineering].

Representatives of Bifidobacterium genus are considered to play many important roles in intestinal homeostasis. On the other hand, their molecular biology and genetics have been poorly studied. In order to broaden our understanding of their health-promoting mechanisms, it is extremely important to possess tools to manipulate them genetically. Another challenging task is to take advantage of genetic engineering technology for designing new probiotic bifidobacteria with unique therapeutic properties. An important step in such work is to isolate and characterize small bifidobacterial plasmids, which can be applied to the construction of cloning vectors. This article presents a review of several pioneering studied devoted to bifidobacterial plasmids and genetic engineering with bifidobacteria. Trends in and prospects of molecular genetics of bifidobacteria are discussed as well.

Search for protein adhesin gene in Bifidobacterium longum genome using surface phage display technology.

A fragment of the nucleotide sequence encoding polypeptide binding to HT-29 epithelial cells was cloned from VMKB44 Bifidobacterium longum genome library using surface phage display technology. Sequencing of this polypeptide consisting of 26 amino acid residues showed that it is an extracellular fragment of a large BL0155 transmembrane protein belonging to the ABC transport protein superfamily. The genes encoding homologues of this protein were detected in genomes of not only bifidobacteria of different species, but also in many other enteric commencals and pathogens.

[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.