[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.

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.

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.

[Sensitivity of corynebacterium diphtheriae strains to antibacterial preparations].

AIM: Study the prevalence and mechanisms of resistance in circulating C. diphtheriae strains. MATERIALS AND METHODS: 664 C. diphtheriae strains isolated in 1987 - 2013 in various regions of Russia and sent to the reference center of Gabrichevsky Moscow Research Institute of Epidemiology and Microbiology were the object of the study. Antibiotic sensitivity of the strains was studied by disk-diffusion and E-test methods using 10 antimicrobial preparations. Nucleotide sequence analysis was carried out by using BLAST program and EMBL/GenBank database. RESULTS: Most of the studied strains turned out to be sensitive to all the antibacterial preparations used. 1.2% of C. diphtheriae strains turned out to be resistant to penicillin and 6.0% had intermediate level of resistance. 0.4 - 0.6% of the strains had intermediate level of resistance to macrolides, and 4.0 - 4.4% were resistant. 2.0% of the strains had multiple resistance. Erm(X)-specific PCR carried out in this study showed that all the C. diphtheriae strains resistant to macrolide antibiotics carry erm(X) gene. CONCLUSION: The results of the study indicate a fairly high level of prevalence for C. diphtheriae strains resistant to antibiotics.

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.

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).

[Molecular mechanisms of induction of innate immunity].

Cellular and molecular mechanisms of congenital immunity at different levels are discussed including single cell expression patterns and intracellular localization of individual TLR, the use of adapter molecules for generation of activation signals in response to microbial and non-microbial pathogens, soluble trap receptors, and intracellular negative regulators.

[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.

[The role and biological significance of toll-like receptors in the anti-infectious resistance of the organism].

Modem conceptions of the role of toll-like receptors (TLR) in the innante immunity mechanisms realization and data on the interaction between TLR and pattern-associated molecular proteins of microbial or endogenic origin are presented in the review.

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.

[Chronic heart failure: structural and microbiological changes in the colon].

AIM: To study microbiocenosis of the parietal layer of the colon and feces, concentrations of endotoxin and proinflammatory cytokines in patients with chronic heart failure (CHF) of different functional classes vs. healthy subjects of the same age. MATERIAL AND METHODS: The trial includes 37 patients with ischemic CHF and 13 healthy volunteers. The examination comprised 6-min walking test, echocardiographic evaluation of the left ventricular ejection fraction, clinical state by a special scale, assay for C-reactive protein, endotoxin, fecal seeding, colonoscopy with biopsy and seeding. RESULTS: Gram-negative flora in the colon and parietal layer occurred in high concentrations correlating with severity of CHF. The examinees with CHF of functional class III-IV had elevated levels of circulating endotoxin and serum C-reactive protein.

[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.

[A molecular-and-genetic analysis of species and strain diversity of bifidobacteria in early childhood].

The species and strain composition of bifidobacteria in 29 children both sexes, aged 8 to 16 months, was studied. Species-specific primers and PCR were used to determine to which species the predominant strains of bifidobacteria, isolated from feces by cultural methods, belonged. Bifidobacteria were found in 28 (96.5%) children; their number was 10.2 +/- 0.7 ECU per a gram of the material. B. longum and B. bifidum were frequent (71.4 and 53.5%, respectively). The level of quantitative detection used in the study also allowed revealing of B. catenulatum (17.9%) and B. breve (14.4%). A high titer of B. dentium was found in one case (3.6%). B. adolescentis and B. angulatum were not found in any patient. The average number of species found in one child was 1.7 +/- 0.7. RAPD-PCR and investigation of plasmid profile were used to determine possible belonging of the isolates to different strains. The average number of strains per one sample was 2.3 +/- 1.2. Nine unique plasmid bifidobacterial strains were isolated from 7 children. In 3 children the intestinal tract was found to be colonized by both plasmid and non-plasmid-carrying strains of one bifidobacterial species.

[Problem of ENT hospital infection].

Five-year monitoring of microbial environment of the ENT department stated its relative qualitative persistence. Circulating in hospitals microbial strains have a direct influence on the course of the infectious process in the patients. Hospital infection in the ENT department replaces the initial pathogen for a polyresistant hospital pathogen or forms a persistent microbial association.