Barbara Sgorbati

Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.)

The present study focused on inhibitory activity of freshly extracted essential oils from three legal (THC<0.2% w/v) hemp varieties (Carmagnola, Fibranova and Futura) on microbial growth. The effect of different sowing times on oil composition and biological activity was also evaluated. Essential oils were distilled and then characterized through the gas chromatography and gas chromatography-mass spectrometry. Thereafter, the oils were compared to standard reagents on a broad range inhibition of microbial growth via minimum inhibitory concentration (MIC) assay. Microbial strains were divided into three groups: i) Gram (+) bacteria, which regard to food-borne pathogens or gastrointestinal bacteria, ii) Gram (-) bacteria and iii) yeasts, both being involved in plant interactions. The results showed that essential oils of industrial hemp can significantly inhibit the microbial growth, to an extent depending on variety and sowing time. It can be concluded that essential oils of industrial hemp, especially those of Futura, may have interesting applications to control spoilage and food-borne pathogens and phytopathogens microorganisms.

Gut health promoting activity of new putative probiotic/protective Lactobacillus spp. strains: a functional study in the small intestinal cell model.

In interaction studies with the host intestine, the use of the appropriate gut functional cell model is essential. Therefore, we examined the protective properties of selected lactobacilli in a newly established intestinal cell model. Bacteria were cocultured with the pig small intestinal epithelial cells (PSIc1) and pig blood monocytes (PoM2) in a functional intestinal cell model. Intercellular intestinal integrity was measured by transepithelial electrical resistance (TER), before and after coculture with selected bacterial strains. All selected bacterial strains showed important gut health promoting activity by: enhancing the intestinal integrity and increasing metabolic activity of intestinal cells. Stimulation of immune response was strain specific. The best stimulants were unidentified lactobacillus strains obtained from fermented food in Africa (PCK87 and 66), followed by Lactobacillus plantarum (PCS26). Their activity was significantly higher (p<0.05) than that of the commercial Lactobacillus casei Shirota strain.

Purification and properties of two fructose-6-phosphate phosphoketolases in Bifidobacterium

Fructose-6-phosphate phosphoketolase was purified from type strains of two species of the genus Bifidobacterium: B. globosum and B. dentium. The first species has a preferred “animal” habitat, like feces of animals and rumen of cattle; the latter is harboured in “human” habitats, like feces and dental caries of man. Two electrophoretic types of phosphoketolase (F6PPK) were previously distinguished and called “animal” and “human” type according to the habitat of the bifid organism. The purified preparations of these two phosphoketolases displayed very different optimum pH range, metal activator and molecular weight; outstanding difference was found in the substrate specificity: the enzyme from B. globosum was able to split xylulose-5-P as well as fructose-6-P, whereas the phosphoketolase from B. dentium appeared to be specific for fructose-6-P.

Electrophoretic types of transaldolase, transketolase, and other enzymes in bifidobacteria

The technique of starch-gel electrophoresis with staining for transaldolase, transketolase, 6-phosphogluconate dehydrogenase, and aldolase, was used to compare 49 representative strains of the genus Bifidobacterium, the deoxyribonucleic acid homology relationships of which were known. The zymograms obtained with fructose-6-phosphate as substrate for staining were also recorded and compared. Parallel experiments were made with spectrophotometric techniques to evaluate the specificity of the staining for transaldolase and transketolase. In each of the enzymatic activities investigated, isozymes were revealed by this technique. Their distribution is discussed.

Demonstration of Phylogenetic Relatedness Among Members of the Genus Bifidobacterium by Means of the Enzyme Transaldolase as an Evolutionary Marker

Antisera prepared against the purified transaldolases of five species of Bifidobacterium were used to establish natural relationships among the 21 species that presently comprise this genus. The degree of phylogenetic relatedness among the respective members of this genus was estimated from the results of qualitative and quantitative immunological procedures in which the enzymes served as an evolutionary marker. The results are presented in the form of a phylogenetic dendrogram.

Occurrence of Bifidobacteriaceae in human hypochlorhydria stomach

Background The human stomach, when healthy, is not a suitable host for microorganisms, but in pathological conditions such as gastritis, when gastric acid secretion is impaired, microbial overgrowth can be observed. Apart from Helicobacter pylori, the composition of microbiota, resident or exogenously introduced during neutral/high pH conditions, has not been investigated thoroughly. Thus, it is possible that Bifidobacteriaceae, important autochthonous and beneficial bacteria of human gastrointestinal microbiota, could over-colonize the stomach of hypochlorhydria patients suffering from autoimmune atrophic gastritis (AAG) or omeprazole-treated (OME) gastritis. This prompted us to characterize the Bifidobacteriaceae in such patients’ gastric microbiota and to study its abnormal colonization. Methods Samples of gastric juices, and antrum and corpus mucosa from 23 hypochlorhydria patients (13 AAG and 10 OME) and from 10 control volunteers with base-line normochlorhydria, were cultivated in Brain Heart Infusion (BHI) and selective Bifidobacterium-Tryptone-Phytone-Yeast extract (Bif-TPY) media. The isolates were characterized by the fructose-6-phosphate phosphoketolase (F6PPK) test, electrophoresis of cellular proteins, the fermentation test, guanine-cytosine% DNA content, and DNA–DNA hybridization. Negative F6PPK isolates were characterized by order-specific polymerase chain reaction (PCR). Results A total of 125 isolates, assigned to the Bifidobacteriaceae family on the basis of their morphology, were obtained from AAG and OME patients, but not from normal subjects. Of these isolates, 55 were assigned to the Bifidobacteriaceae family on the basis of their fructose-6-phosphoketolase (PPK) activity, PPK being the key taxonomic enzyme of this family. The remaining 70 isolates, which were PPK-negative, were attributed to the Actinomycetales order following specific primer PCR analysis. We observed a significantly higher abundance of Bifidobacteriaceae (Bifidobacterium dentium, Scardovia inopinata, and Parascardovia denticolens) in OME group than the AAG group. Furthermore, the Actinomycetales distribution was homogeneous for both hypochlorhydria patient groups. Conclusions This study suggests that the Bifidobacteriaceae species, typically found in the oral cavity, readily colonizes the hypochlorhydria stomach of OME patients. The clinical relevance and the mechanism underlying this Bifidobacteriaceae presence in OME gastritis requires further functional studies.

Preliminary quantification of immunological relationships among the transaldolases of the genus Bifidobacterium

The immunological relatedness among the transaldolases (dihydroxyacetone transferase, E.C. 2.2.1.2) of twenty species of the genus Bifidobacterium has been tested by the microcomplement fixation method, using B. thermophilum (B. ruminale) RU326 (=ATCC 25866), B. cuniculi RA93(=ATCC 27916) and B. ‘minimum’ (DNA homology group) F392 (=ATCC 27538) as references. Based on the serological relationships of the transaldolases, expressed either as indices of dissimilarity or as immunological distances, the twenty species of the genus Bifidobacterium were arranged into clusters. These clusters generally coincided with the immunological groups obtained previously by the immunodiffusion method (Sgorbati and Scardovi, 1979).

Lactobacillus salivarius and L-gasseri down-regulate Aggregatibacter actinomycetemcomitans exotoxins expression

Beneficial microbes, such as lactobacilli establish a symbiosis with the host and confer health-associated effects, by limiting the growth of indigenous pathogens and challenging microbes introduced by altered foods. Nevertheless, there is scarce information on the effects of beneficial microbes on the virulence properties of bacterial species associated with oral diseases, such as periodontitis. Aggregatibacter actinomycetemcomitans is a Gram-negative species highly implicated in the etiology of localized aggressive periodontitis. The objective of this study was to investigate the effect of lactobacilli on the expression of the two major virulence factors of A. actinomycetemcomitans. Lactobacillus salivarius and L. gasseri were selected as beneficial species. The gene expressions of leukotoxin (LtxA) and cytolethal distending toxin (CdtB) by A. actinomycetemcomitans were analyzed in response to challenge by lactobacilli cell-free supernatants. Neither lactobacilli affected the growth, but strongly attenuated the expressions of both CdtB and LtxA in the two A. actinomycetemcomitans strains tested. This reduction of the expression of these two exotoxins was time-dependent. These fundamental findings may indicate that lactobacilli can reduce the virulence of putative opportunistic oral pathogens, and may provide insights to future therapeutic approaches for the respective diseases.

Immunological relationships among transaldolases in the genus Bifidobacterium

Antisera were prepared against electrophoretically homogeneous transaldolase (dihydroxyacetone transferase, E.C. 2.2.1.2.) of Bifidobacterium thermophilum (B. ruminale) RU326 (ATCC 25866), B. cuniculi RA93 (ATCC 27916) and B. ‘minimum’ (homology group) F392 (ATCC 27538).Crude extracts of eighty six strains previously assigned to twenty one species of the genus Bifidobacterium on the basis of deoxyribonucleic acid (DNA) homology (DNA-DNA hybridization), were compared by double diffusion tests on Ouchterlony plates. Eight groups of identical antigenic specificity were recognized. By analysis of the spur formation, the groups of identical specificity were arranged in preliminary sequences of decreasing similarity to each of the three homologous transaldolases used as reference point. The relationships between immunological data and the genetic similarity among the species of the genus measured by means of DNA-DNA hybridization were discussed together with some relevant points of bifidal ecology.

Bifidobacterium. eulemuris sp. nov. isolated from the faeces of the black lemur (Eulemur macaco).

Forty-three strains of bifidobacteria were isolated from the faeces of two adult black lemurs, Eulemur macaco. Thirty-four were identified as Bifidobacterium lemurum, recently described in Lemur catta. The nine remaining isolates were Gram-positive-staining, non-spore-forming, fructose-6-phosphate phosphoketolase-positive, microaerophilic, irregular rod-shaped bacteria that often presented Y- or V-shaped cells. Typing techniques revealed that these isolates were nearly identical, and strain LMM_E3T was chosen as a representative and characterized further. Phylogenetic analysis based on 16S rRNA gene sequences clustered this isolate inside the genus Bifidobacterium and showed the highest levels of sequence similarity with B. lemurum DSM 28807T (99.3 %), with Bifidobacterium pullorum LMG 21816T and Bifidobacterium longum subsp. infantis ATCC 15697T (96.4 and 96.3 %, respectively) as the next most similar strains. The hsp60 gene sequence of strain LMM_E3T showed the highest similarity to that of Bifidobacterium stellenboschense DSM 23968T (93.3 %), and 91.0 % similarity to that of the type strain of B. lemurum. DNA-DNA reassociation with the closest neighbour B. lemurum DSM 28807T was found to be 65.4 %. The DNA G+C content was 62.3 mol%. Strain LMM_E3T showed a peptidoglycan structure that has not been detected in bifidobacteria so far: A3α l-Lys-l-Ser-l-Thr-l-Ala. Based on the phylogenetic, genotypic and phenotypic data, strain LMM_E3T represents a novel species within the genus Bifidobacterium, for which the name Bifidobacterium eulemuris sp. nov. is proposed; the type strain is LMM_E3T ( = DSM 100216T = JCM 30801T).