Aggregation and Adhesion Activity of Lactobacilli Isolated from Fermented Products In Vitro and In Vivo: a Potential Probiotic Strain

Approximately 25 strains of lactobacilli isolated from different dairy products and fermented vegetables were screened according to their possibility to show the high auto-aggregation and co-aggregation. The strains Lactobacillus helveticus INRA-2010-H11, Lactobacillus rhamnosus INA-5.1, and Lactobacillus acidophilus JM-2012 were determined to have the high auto-aggregation (approximately 73, 46, and 70.5% correspondingly). A high co-aggregation capacity (75.53%) for strains INRA-2010-H11 and JM-2012 was shown. The adhesion degree of INRA-2010-H11 on the surface of buccal epithelial cells was 88.23%. The study of INRA-2010-H11, JM-2012, and both strains’ mixture (1:1) adhesion capacity on the surface of epithelial HeLa cells revealed the adhesion of 1.1 × 10⁶, 6.3 × 10⁴, and 2.3 × 10⁵ CFU, respectively, from starter amount of CFU 10⁷ and 10⁸ for both strains. In vivo experiments of LAB adhesion in gastrointestinal tract of mouse revealed the presence of 2.5 × 10⁹, 1.2 × 10⁹, and 1.5 × 10⁹ CFU of LAB in control and groups of mouse, fed by INRA-2010-H11 and mixture, respectively. Feeding by investigated lactobacilli was suggested to lead to microbiota biodiversity reduction in small intestine and colon and its augmentation in stomach. Thus, INRA-2010-H11 demonstrated a high aggregation and adhesion activity so it has the potential as a good probiotic strain.     

Effect of Sodium Taurocholate on the In Vitro Growth of Lactobacilli

Abstract The effect on growth of a conjugated bile salt (sodium taurocholate) at physiological concentration was determined using cultures of Lactobacillus strains of murine origin. The bile salt stimulated the growth of one strain, did not affect the growth of another, but inhibited the growth of strains that produced relatively large amounts of the enzyme bile salt hydrolase. Comparison of the growth of isogenic strains that differed in the ability to produce bile salt hydrolase demonstrated that inhibition of growth was due to the accumulation of cholic acid in the culture medium as a result of the enzyme activity. Received: 15 January 1996; Revised: 26 March 1996; Accepted: 29 March 1996     

Sex Differentiation of Avian Gonads In Vitro

The analysis of avian sex differentiation in vitro has beenlimited to the following problems: morphological sex differentiationof gonads cultured in vitro; analysis of the chemical natureof the hormonal secretion; differentiation of germ cells inrelation to their somatic environment. Morphological sex differentiationof avian gonads occurs in vitro. Differentiated gonads of thechick embryo carry out biosynthesis of sex hormones from severalradioactive precursors. Female gonads in particular synthesizeestrogens while male gonads synthesize testosterone. Some experimentshave given evidence of estrogen synthesis by undifferentiatedfemale gonads. Embryonic gonads of quail, like those of chick,are able to synthesize sex steroids from radioactive precursors.However, in the quail and mainly in the testes, a delayed appearanceand a lower activity of the enzyme system 3ß-HSDHs-4-isomerase was found. Histoenzymological results corroboratethe biochemical ones. Combination of culture and grafting experimentshave shown that male germ cells when they are forced into femaledifferentiation by early colonization of a female gonad degenerateafter entering the premeiotic stage. The reasons for this delayedfailure of sex differentiation of "male oocytes" have certainlyto be searched for at the level of perturbation in the mechanismsof meiosis.     

In vitro adhesion specificity of indigenous Lactobacilli within the avian intestinal tract

In vitro adherence of Lactobacillus strains to cell and tissue types along the chicken alimentary tract and to ileal mucus were determined. Fresh isolates from chickens adhered to the epithelium of crop and, in a strain-dependent manner, to follicle-associated epithelium and the apical surfaces of mature enterocytes of intestinal villi. No adherence to the apical surfaces of undifferentiated enterocytes, the mucus-producing goblet cells, or the ileal mucus was detected.     

An In Vitro Study on Bacterial Growth Interactions and Intestinal Epithelial Cell Adhesion Characteristics of Probiotic Combinations

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.     

Effects of Hydrogel/Silver Coatings on In Vitro Adhesion to Catheters of Bacteria Associated with Urinary Tract Infections

Sections of sterile all-silicone-, hydrogel/silver-all-silicone-, and hydrogel/silver-latex-Foley urinary catheters were exposed to suspensions of bacteria and Candida albicans associated with urinary tract infections. The adhesion of these microorganisms to the catheters was determined with a radiolabel–cell procedure and scanning electron microscopy. Anomalous data with the radiolabel procedure were produced with the hydrogel/silver-latex catheters for certain species. These aberrant data were related to adhesion on the untreated cut ends of the latex catheter. Radiolabel-cell-adhesion procedures that involve sections of coated materials may need to be supplemented with additional procedures such as scanning electron microscopy for valid interpretations of the data. Adhesion to the hydrogel/silver catheters by both Gram-positive- and Gram-negative bacteria most commonly associated with nosocomial urinary tract infections, including a strain of Pseudomonas aeruginosa noted for its superior adhesion capacity, was significantly lower than the adhesion to the control all-silicone catheter. Received: 21 January 2000 / Accepted: 26 February 2000     

In Vitro Development of Exoerythrocytic Forms of Plasmodium Gallinaceum Sporozoites In Avian Macrophages

ABSTRACT Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. the host cells were macrophage precursors from chicken bone marrow. At various times after introduction of Sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). the time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until Ac end of the primary exoerythrocytic schizogony.     

Bile Salt Hydrolase (Bsh) Activity Screening of Lactobacilli: In Vitro Selection of Indigenous Lactobacillus Strains with Potential Bile Salt Hydrolysing and Cholesterol-Lowering Ability

The bile salt hydrolase (Bsh) activity of probiotic bacterium residing in gastrointestinal tract has often being associated with its cholesterol-lowering effects. Hence, Bsh activity was explored in this study as the criterion for the selection of most potential Bsh-active and cholesterol-lowering indigenous Lactobacillus strains. Forty lactobacilli were adjudged Bsh active after a preliminary screening of 102 lactobacilli and occurrence of Bsh activity correlated well with their natural habitats. Of the 40 shortlisted lactobacilli, fifteen putative Lactobacillus strains were selected and further tested for their comparative Bsh activity. In the end, indigenous Lactobacillus plantarum strains Lp91 and Lp21 were emerged as the promising Bsh-active lactobacilli with their substrate preference inclined more towards glycocholate than other bile acid amino conjugates. In addition, strains Lp91 and Lp21 also exhibited significantly high bile salt deconjugation, cholesterol assimilation and cholesterol co-precipitation ability in vitro. In conclusion, indigenous L. plantarum strains Lp91 and Lp21 may be the promising candidate probiotics to elucidate the ecological significance of probiotic Bsh activity in vivo.     

Inhibition of Giardia intestinalis by Extracellular Factors from Lactobacilli: an In Vitro Study

The aim of the present work was to evaluate the effect of spent culture supernatants of different strains of lactobacilli on giardia trophozoites. The growth of Giardia intestinalis strain WB, as well as the attachment to the human intestinal epithelial cell line Caco-2, was evaluated by using proliferation and adhesion assays with radiolabeled parasites. In addition, scanning electron microscopy and flow cytometric analysis were performed. The effect of spent culture supernatants from lactobacilli was strain dependent. Lactobacillus johnsonii La1 significantly inhibited the proliferation of G. intestinalis trophozoites. Although the effect was strongly pH dependent, it was not simply due to lactic acid. According to flow cytometric analysis, trophozoites were arrested in G₁ phase but neither significant necrosis nor apoptosis could be detected. Bacterial cells or their spent culture supernatants were unable to modify trophozoite attachment to Caco-2 cells. However, trophozoites treated with spent culture supernatants had little, if any, proliferative capacity. These results suggest that La1 produces some substance(s) able to inhibit proliferation of Giardia trophozoites. Partial characterization of the factors involved in the antigiardiasic action showed that they have a low molecular mass and are inactivated by heating. On this basis, it seems worthwhile to explore how colonization of the proximal small bowel with these lactic acid bacteria could interfere with giardiasis in vivo.     

Microbial Interference Between Indigenous Yeast and Lactobacilli in the Rodent Stomach

Indigenous yeasts grow in layers in the mucus on the secreting epithelium of the stomachs of some strains of rats and mice raised under conventional conditions. Likewise, indigenous lactobacilli appear in layers on the nonsecreting epithelium of the stomachs of rats and mice. The two microbial layers can coexist in the same animals. When I gave such rodents penicillin solution in the place of drinking water, the lactobacilli disappeared, and the yeast from the secreting epithelium colonized the nonsecreting epithelium within 24 hr. The yeast remained in layers on the nonsecreting, as well as the secreting epithelium, as long as penicillin was administered. There is no inflammatory reaction or any sign that the yeast invaded below the keratin layer. When the penicillin treatment was discontinued, within 5 to 8 days the indigenous lactobacilli again colonized the nonsecreting epithelium. Concomitantly the yeast was displaced from the keratinized tissue and once more could be found only on the secreting epithelium. Only 2 days were required, however, for the bacteria to recolonize the keratin layer and displace the yeast when the mice were given indigenous lactobacilli in pure culture immediately after the penicillin treatment was discontinued. The lactobacilli must displace the yeast from the nonsecreting epithelium by interfering either with multiplication of the yeast on the tissue or with attachment of the yeast cells to the keratin layer. This interference must proceed continuously during normal life since the yeast never populates the nonsecreting epithelium as long as the lactobacilli are present.     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Antioxidant Lactobacilli Could Protect Gingival Fibroblasts Against Hydrogen Peroxide: A Preliminary In Vitro Study

Oxidative stress and tissue destruction are at the heart of periodontal diseases. The dental research area is geared toward the prevention of free radicals by nutrient antioxidants. Lactic acid bacteria (LAB) have recently attracted attention in alternative dental therapies. We aimed at highlighting the antioxidative property of Lactobacilli and Bifidobacterium strains and at determining their protective effect on gingival fibroblasts (GFs). Two Lactobacilli and 2 Bifidobacterium strains were screened for their exopolysaccharide (EPSs) production. Antioxidative assays were conducted by spectrophotometer analysis. Resistance to different concentrations of hydrogen peroxide (H₂O₂) was determined by the serial dilution technique. The protective effect of strains on GFs on hydrogen peroxide exposure was also examined by a new trypan blue exclusion assay method. Bifidobacterium breve A28 showed the highest EPS production (122 mg/l) and remarkable antioxidant activity, which were demonstrated by its ability to scavenge 72 % α,α-diphenyl-1-picrylhydrazyl free radical and chelate 88 % of iron ion, respectively. Inhibition of lipid peroxidation was determined as 71 % for the A28 strain. We suggest that LAB with antioxidative activity could be a good natural therapy agent for periodontal disorders.     

In Vitro Activity of Lincomycin and Spectinomycin Against Serotypes of Avian Mycoplasma

Twenty strains of avian mycoplasma, representing 12 serotypes, were tested in vitro for their susceptibility to the action of lincomycin and spectinomycin alone and in combination. They varied in their sensitivity pattern. The ranges of minimal inhibitory concentration were 1 to 20 mug/ml for lincomycin or spectinomycin alone and 0.5/1 to 3/6 mug/ml for the lincomycin and spectinomycin combination. The ranges of minimal lethal concentration were greater with either single antibiotic than with the antibiotic combination. The amount of each antibiotic required to achieve mycoplasmacidal action of the relatively resistant strains was less with the antibiotic combination than with the single antibiotics.     

Circadian Rhythms In the Epithelial Cells and the Pericryptal Fibroblast Sheath In Three Different Sites In the Murine Intestinal Tract

Variations in percentage labelling (LI) and mitotic activity (mitoses per crypt) have been studied over a 24-hr period in the epithelial cells and pericryptal fibroblast sheath (PCFS) of the small intestine, caecum, and colon of the mouse. All three tissues displayed clear, synchronized circadian rhythms in DNA-synthetic activity in both the epithelial cells and PCFS. Peak values were coincident within a tissue, but staggered between tissues. In the epithelial cells, peak mitotic values were found between 3 and 6 hr after the peak LI values. the low level of mitotic activity in the PCFS appeared to be synchronized with the rhythms in the adjacent epithelia. the epithelial cells of the lowest crypt third displayed the clearest circadian rhythms. However, the PCFS cells at all levels produced similar curves. A craniocaudal wave of proliferative activity is proposed.     

猪源乳酸杆菌对鼠伤寒沙门氏菌DT104在猪肠道上皮细胞IPEC-J2上粘附的影响

采用猪肠道上皮细胞株IPEC-J2体外培养模型,考察9株猪源乳酸杆菌对IPEC-J2细胞的粘附特性,以及对鼠伤寒沙门氏菌DT104粘附的竞争、排斥、置换和抗侵袭作用.结果显示,9株乳酸杆菌均能粘附IPEC-J2细胞,粘附率在0.1%～10%之间,具有菌株特异性和浓度效应.乳酸杆菌和沙门氏菌同时加入细胞培养,能竞争性抑制沙门氏菌的粘附,并具有浓度效应,高浓度(109 CFU/mL)添加K30、K67和K16时,抑制率可达80%以上.乳酸杆菌预处理细胞后再加入沙门氏菌,高浓度乳酸杆菌可降低沙门氏菌粘附率40%～70%,而中浓度(108 CFU/mL)乳酸杆菌能抑制23%～33%沙门氏菌对细胞的侵入.但是,只有高浓度添加乳酸杆菌能置换已经粘附的沙门氏菌,置换率在12%～84%之间.该结果为临床上筛选乳酸杆菌,有效防治猪沙门氏菌病提供了一条新的途径.