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.

Correction to: Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species.

In the published article, the co-author Abdelmoneim Abdalla's affiliation has been published incompletely. The additional affiliation is given below.

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.

Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species.

Interest in, and use of, bifidobacteria as a probiotic delivered in functional foods has increased dramatically in recent years. As a result of their anaerobic nature, oxidative stress can pose a major challenge to maintaining viability of bifidobacteria during functional food storage. To better understand the oxidative stress response in two industrially important bifidobacteria species, we examined the response of three strains of B. longum and three strains of B. animalis subsp. lactis to hydrogen peroxide (H2O2). Each strain was exposed to a range of H2O2 concentrations (0-10 mM) to evaluate and compare intrinsic resistance to H2O2. Next, strains were tested for the presence of an inducible oxidative stress response by exposure to a sublethal H2O2 concentration for 20 or 60 min followed by challenge at a lethal H2O2 concentration. Results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H2O2 resistance of all strains tested and B. animalis subsp. lactis BL-04 had the highest resistance among B. lactis strains. Inducible H2O2 resistance was detected in four strains, B. longum NCC2705, B. longum D2957, B. lactis RH-1, and B. lactis BL-04. Other strains showed either no difference or increased sensitivity to H2O2 after induction treatments. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific in B. longum and B. lactis and suggest that for some strains, sublethal H2O2 treatments might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.

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.

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.

Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and alpha-galactosidase as selectable markers.

AIMS: We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumalpha-galactosidase gene (aglL) as selectable markers. METHODS AND RESULTS: The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC-), the resulting vector, pSUW611 (3.9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0.2% loss per generation). The second vector, pSUW711 (5.1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48-72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. CONCLUSIONS: The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis.

[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.

[Effect of the lavage of the digestive tract on microflora in patients with polyps in the large intestine]. / Vliianie lavazha pishchevaritel'nogo trakta na mikrofloru u bol'nykh s polipami v tolstoi kishke.

The microbial status of the intestine and the influence of lavage with polyethylene glycol and balanced electrolyte solution (PEG + E), used in the process of the preparation of patients to polypectomy, on this status were evaluated. The study of microflora was made before oral lavage after, and 48-72 hours later its completion. For control, a group of healthy volunteers, also subjected to oral lavage with PEG + E, was used. The lavage of the digestive tract with PEG + E led to a sharp change in the microbial status in both groups. Some microorganisms, previously absent in the intestine, were found after lavage. The processes of the restoration of intestinal microflora after lavage in healthy volunteers and in patients with polyps had certain differences. In healthy volunteers intestinal microflora was completely restored, and even improved, 48-72 hours after lavage with PEG + E, while at the expiration of this time intestinal microflora in the patients with polyps could be characterized as dysbiotic.

[Qualitative composition of the normal intestinal microflora in individuals from the various age groups]. / Kachestvennyi sostav normal'noi mikroflory kishechnika u lits razlichnykh vozrastnykh grupp.

The study of the microflora of the large intestine in healthy adult volunteers of different age groups (25-36, 55-68 and 88-94 years old), living in Switzerland, has been carried out. As revealed by the analysis of the result obtained in this study, normal intestinal microflora in adults has different qualitative and quantitative characteristics at different periods of their life. The greatest diversity of intestinal microflora is observed at a mature age (55-68 years old), while the poorest microflora is observed in people more advanced in age. The amount of microorganisms inhabiting the intestine reaches its maximum in people of mature age and is low in elderly people. Changes in normal intestinal microflora are probaly linked with morpho-functional transformations in the host body at different periods of life. It is expedient to work out the criteria of norm for the microflora of different age groups with a view to use these criteria for the evaluation of the microbial status of the intestine in persons of different age.

[Intestinal microecology of the adult population of Mongolia, Switzerland, and Russia]. / Mikroékologiia kishechnika vzroslogo naseleniia Mongolii, Shveitsarii i Rossii.

The study of intestinal microflora was made in clinically healthy young adults living in rural areas of Mongolia, in Russia (Moscow) and in Switzerland, as well as in Swiss citizens of elderly age groups (55-68 and 87-94 years). Essential differences in the quantitative and qualitative characteristics of intestinal microflora both in the inhabitants of different countries as well as in people belonging to different age groups in the same country were established. The results of the study demonstrated the expediency of working out the criteria of the norm for intestinal microflora both for the population of different countries and for people living in the same country, but belonging to different age groups.

[Effect of bifidobacteria and lactobacillus and antibiotics in combination with common gnotobiological isolation on the survival of mice with acute radiation sickness]. / Vliianie na vyzhivaemost' myshei pri ostroi luchevoi bolezni bifido- i laktobakterii i antibiotikov v komplekse s obshchei gnotobiologicheskoi izoliatsiei.

The influence of the combined use of bacterial preparations (Bifidobacterium and Lactobacillus) and antibiotics (ciprofloxacin, lomefloxacin and amikacin) on the survival rate of irradiated mice placed under the conditions of general gnotobiological isolation was studied. Bacterial strains used in combination with quinolones (ciprofloxacin, lomefloxacin) significantly increased the mean survival time of the animals (p < 0.05) when introduced in a dose of 1.0 x 10(9) microbial cells per mouse on days 1, 3, 5, 7, 9, 11, 13 and 15 after irradiation. At the same time a short course of treatment with bacterial preparations (two injections on days 5 and 7 after irradiation) proved to be insufficient for increasing the survival rate of the animals. The mean survival time of the irradiated mice was higher after the use of bacterial preparations in combination with lomefloxacin or ciprofloxacin than after their use with amikacin.

[The primary screening of bifidobacteria and lactobacilli strains to develop effective probiotic preparations based on them]. / Pervichnyi skrining shtammov bifidobaterii i laktobakterii s tsel'iu razrabotki na ikh osnove éffektivnykh preparatov-probiotikov.

10 Bifidobacterium strains and 10 Lactobacillus strains were studied for their antagonistic activity with respect to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and for their sensitivity to antibiotics, widely used in clinical practice. L. acidophilus strain 5/4, L. acidophilus strain 18/4, B. adolescentis strain UX, B. longum strain 44 exhibited the highest antagonistic activity and the highest degree of antibiotic resistance. The restriction analysis of the chromosomal DNA of these strains was then made and their plasmid content was studied, making it possible to recognize these strains in future in the course of in vivo experiments.

[The antagonistic activity of bifidobacteria in vitro and in vivo studied by using gnotobiological technology]. / Izuchenie antagonisticheskoi aktivnosti bifidobakterii in vitro i in vivo s izpol'zovaniem gnotobiologicheskoi tekhnologii.

The antagonistic activity of 4 strains of bifidobacteria (B. adolescentis 2 F1, B. longum Z4, B. breve R2 and B. bifidum G1), isolated from the vagina of healthy females of the reproductive age, with respect to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Gardnerella vaginalis were studied in vitro and in vivo. The in vitro experiments revealed that all above-mentioned bifidobacteria were capable of inhibiting the growth of all indicator bacterial strains. Still of all the bifidobacteria under study had different levels of activity. B. adolescentis strain 2 F1 exhibited the highest inhibiting activity in vitro. In contrast to in vitro experiments, in vivo experiments with B. breve R2 demonstrated its high antagonistic activity with respect to E. coli. The data thus obtained indicate that in the study of antagonistic activity the use of the in vivo model as also expedient, for it is mainly in vivo that probiotic preparations show their activity.

[The vaginal Bifidobacterium flora in women of reproductive age]. / Izuchenie bifidoflory vlagalishcha u zhebshchin reproduktivnogo vozrasta.

The composition of vaginal bifidoflora in 56 clinically healthy women of reproductive age was studied. The study revealed that four species of bifidobacteria, viz. Bifidobacterium bifidum, B. breve, B. adolescentis 2 and B. longum, dominated in the composition of this bifidobacterial population. Nine out of 11 isolated strains were found to be capable of inhibiting indicator microorganisms Staphylococcus aureus and Enterococcus faecalis when tested in vitro; in addition, strains B. adolescentis 2 F1, B. bifidum G1, B. breve P2 and B. longum Z4 inhibited Klebsiella ozaenae, Pseudomonas aeruginosa, Escherichia coli and were also active acid producers. Three of these 4 bifidobacterial strains were capable of adhesion to vaginal epitheliocytes, while B. bifidum G1 was practically incapable of adherence to these cells, similarly to B. bifidum strain 791 of intestinal origin. In addition, the spectra of antibiotic susceptibility varied from strain to strain, but all bifidobacterial strains were susceptible to benzylpenicillin and resistant to lomefloxacin, most of them being also resistant to cyprofloxacin and gentamicin. Thus the data presented in this work are indicative of the possibility and advantages of using bifidobacterial strains belonging to this ecological niche as probiotics for the correction of the microflora of the urogenital tract in females.

Therapeutic use of an antibiotic-resistant Bifidobacterium preparation in men exposed to high-dose gamma-irradiation.

Five men received high-dose, uneven, whole-body gamma-irradiation by accidental exposure to an unshielded 137Cs source. Analysis of the faeces 9-12 days post-irradiation showed low numbers of anaerobes and high counts of enterobacteria and staphylococci in four of the patients and total viable counts of < 10(3)/g in one. All five were treated with systemic ampicillin and gentamicin and oral nystatin commencing 4-7 days after irradiation. Three of the patients were also treated orally with a suspension of an antibiotic-resistant strain of Bifidobacterium longum for 30 days commencing 10-12 days post-irradiation. At 3 weeks post-irradiation, B. longum had appeared in their faecal flora and total anaerobe counts exceeded those of facultative and obligate aerobes. At 4 weeks and 5-7 weeks post-irradiation, this normalisation of the faecal flora continued. In contrast, in the two patients who received a placebo the faecal flora was dominated by enterobacteria (Klebsiella, Enterobacter and Serratia spp.) showing multiple antibiotic resistance 3 weeks post-irradiation. These potential opportunist pathogens were not isolated from the B. longum-treated group. Only one patient in the control group survived beyond 3 weeks; he continued to show high faecal counts of enterobacteria and staphylococci and low counts of obligate anaerobes. 'Probiotic' treatment with this antibiotic-resistant strain of B. longum may be of benefit in the treatment of radiation sickness, aiding normalisation of the faecal flora and inhibiting colonisation and overgrowth with opportunist pathogens.

[Rational approach to correction of intestinal microflora]. / Ratsional'nye podkhody k probleme korrektsii mikroflory kishechnika.

This paper reviews the present notions of the mechanisms of probiotics' action and analyzes selective approaches to correcting the intestinal microflora, such as the use of antibiotic-resistant and highly-adhesive probiotics, treatment with autostrains of lactobacilli and bifidobacteria, and the application of fermented-milk probiotics. Methods for optimization of the intestinal microflora in the newborns by using the maternal strains of bifidobacteria and the drug Zlemik that contains highly-adhesive lactobacilli are discussed. It is shown that parameters of immunotropic activity and involvement in the bacteriocin-mediated interactions may be used to design new probiotics. In future, the application of gene engineering methods will aid in designing a new generation of probiotics with predicted biological properties.

[The effect of Lactobacillus acidophilus "Solco" on the immunological indices of totally decontaminated mice under complete gnotobiological isolation]. / Vliianie Lactobacillus acidophilus "Solco" na immunologicheskie pokazateli total'no dekontaminirovannykh myshei v usloviiakh obshchei gnotobiologicheskoi izoliatsii.

The possibility of stimulating the immunity of totally decontaminated mice, kept isolated under germ-free conditions, with the use of killed L. acidophilus Solco strains has been studied. As revealed in this study, the oral and intraperitoneal administration of strains O1 and O6 to mice leads to a significant increase in the content of immunoglobulin-synthesizing cells in the jejunal lamina propria of the animals. The oral administration of L. acidophilus Solco strain O6 and the intraperitoneal injection of L. acidophilus Solco strain O1 have been found to lead to a significant rise in the level of luminol-dependent chemiluminescence of mouse peritoneal macrophages. The total decontamination of mice induces the development of secondary immunodeficiency which influences the effectiveness of immunostimulating agents.

[Study of the action of immunomodulator T-activin on electric properties of plasma membranes of thymus cells by the use of fluorescent probes]. / Izuchenie deistviia immunomoduliatora T-aktivina na élektricheskie svoistva plasmaticheskoi membrany kletok timusa s pomoshch'iu fliuorestsentnykh zondov.

T-activin alters the electric properties of plasmatic membranes of the T-activin treated suspension of mouse (CBAX X057B1) F1 thymic cells. These alterations were registered by means of negative charged fluorescent probe l-anilino-naphthalene-8-sulfonate (ANS) and positive charged 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM) by measurement of their fluorescence in the individual thymic cell. Apparently, T-activin leads to depolarization of transmembrane potential on plasmatic membrane of thymic cells. It seems that action of T-activin is similar to the effect of polycations, which alter cells ionic streams during lymphocyte activation. However, there is a version, that T-activin acts like neuraminidase, but it is activity less expressed.