Partial characterization of bifidobacterium breve C50 cell-free whey compounds inducing modifications to the intestinal microflora.

Cell-free whey obtained from milk fermented with Bifidobacterium breve C50 (Bb C50) has been shown to modify the intestinal flora in humans and mice. Previous work showed that no antibiotic-like or barrier effect due to overgrowing bifidobacteria was implied in the microbial modifications. The present study was focused on characterizing the compounds and mechanisms involved. Protein, sugar, and enzymatic profiles of Bb C50 whey were therefore determined and compared to those of a whey unable to modify the intestinal flora of humans and mice. No remarkable difference was noted except for a higher lactosidase activity in Bb C50 whey. Various physical treatments were then applied to fractions of Bb C50 whey. Activity was assessed in C3H mice by analyzing changes in the intestinal flora balance throughout a 15-d administration of each treated whey. Heating at 80 degrees C and aerobic storage for 2 wk completely abolished Bb C50 whey activities. In contrast, the addition of a reducing agent (cysteine hydrochloride), either at the beginning of a 15-d aerobic storage or prior to administration, as well as preserved these activities. Susceptibility to heating and oxidation suggested that an enzyme might play a role in the induced microbial changes. Since the Bb C50 lactosidase was partly inactivated by the oxidative treatment, it could support the in vivo activity. The enzyme might reach the intestinal lumen and partly degrade substrates, such as mucins, usually used by the intestinal flora. The released molecules might then favor the development of a new microbial balance.

Bacterial microleakage of Cavit, IRM, TERM, and Fermit: a 21-day in vitro study.

The aim of our study was to evaluate the leakage of four cements (Cavit, IRM, TERM, and Fermit) using a two-compartment model system and Streptococcus sanguis as bacterial marker. Access cavities in premolars were filled with cement and the teeth immersed in culture medium in the model system. Half of the teeth were thermocycled on day 2. Bacterial percolation into the upper compartment was measured at regular intervals (days 2, 7, 14, and 21). Cement thickness was measured at the end of the study. In the nonthermocycled group, Cavit was more leakproof than the other cements at day 2 (p = 0.011), than TERM and IRM at day 7 (p = 0.043). Fermit was more leakproof than IRM at day 7 (p = 0.043). In the thermocycled group, Cavit was more leakproof than the other cements at day 7 (p = 0.041). Thermocycling did not significantly affect leakage. Cement thickness averaged 4.1 mm and did not significantly affect leakage. These results should be considered when using cements as temporary fillings.

Cell-free whey from milk fermented with Bifidobacterium breve C50 used to modify the colonic microflora of healthy subjects.

The ingestion of viable bacteria is thought to be required to modify intestinal microflora. In the present study, the effects on fecal flora of consumption of cell-free concentrated whey from milk that had been fermented with Bifidobacterium breve C50 was tested using 10 healthy human volunteers. Results were compared with effects of a commercial milk formula that had been fermented with Streptococcus thermophilus and B. breve C50 and given to 10 control subjects. Nitroreductase and beta-glucuronidase activities were assessed as risk indexes for colon carcinogenesis, and beta-galactosidase was measured as an indicator of the fermentation capacity of the colonic flora. Fecal excretion of Bacteroides fragilis, Clostridium perfringens, and clostridial spores decreased after 7 d of consumption of either preparation; however, counts of bifidobacteria only increased after intake of B. breve whey. Fecal pH was reduced from 7.1 +/- 0.2 to 6.6 +/- 0.3 after intake of whey that had been fermented with Bif. breve. Fecal nitroreductase and beta-glucuronidase significantly decreased, and beta-galactosidase activity increased, after consumption of either preparation. The results indicate that ingestion of viable bifidobacteria was not required to modify intestinal flora of humans. Repression of B. fragilis and clostridia seems to be independent of colonic bifidobacterial overgrowth in humans.

Cell-free wheys from bifidobacteria fermented milks exert a regulatory effect on the intestinal microflora of mice and humans.

Bacteroides fragilis and clostridia are normally present in the human colon but they may exert pathogenic effects when the homeostasis is upset following various forms of stress. One approach to preventing gastrointestinal disorders is to use bifidobacteria fermented milk. It has been suggested that the efficacy of such a product is related to abiotic compounds produced during milk fermentation. Experiments reported in this paper attempt to check this theory. Six whey retentates were prepared by fermenting cow's milk with six human strains of Bifidobacterium breve and acetic and lactic acids were eliminated by ultrafiltration. Their ability to reduce intestinal clostridial carriage was assessed in C3H mice. Only one whey retentate led to a decrease in clostridia, bacilli, B. fragilis and fecal pH and to an increase in bifidobacteria. Assays in ten human volunteers resulted in similar changes in fecal flora and fecal pH within 7 days of whey retentate intake (30 mL/day). No antibiotic-like effect was demonstrated in vitro. Compounds involved in microflora regulation were located in two ultrafiltrated fractions (30-100 and 100-300 kDa). Both fractions contained mainly low molecular weight glycoproteins (20-40 kDa) and two high molecular weight glycoproteins (121, 211 kDa) that were almost undetectable in the inactive 10-30 kDa fraction.

Bifidobacteria and human health: regulatory effect of indigenous bifidobacteria on Escherichia coli intestinal colonization.

Bifidobacteria are assumed to exert colonization resistance to enteric pathogens. We associated C3H germfree mice with either Bifidobacterium longum or Escherichia coli or both strains and studied how they settled in the gut and the lymphoid organs as well as their effect on mucus composition. Within 24 hE. coli colonized the gut of germfree or B. longum ex-germfree mice. In contrast,B. longum was established in the intestine of E. coli ex-germfree mice only 1 month after inoculation whereas it colonized the germfree gut within 24 h. Although B. longum did not exert colonization resistance to E. coli, the establishing of bifidobacteria in the gut partly prevented changes in the E. coli cell wall. After colonization of the germfree or B. longum mono-associated mice, E. coli lipopolysaccharide exhibited a higher concentration of Kdo and the O-antigen side chain disappeared. A reduction in Kdo content was observed within 1 month in E. coli-B. longum diassociated mice whereas it remained at a high level in E. coli mono-associated mice. Association in a second step with B. longum led to Kdo reduction. Changes in E. coli LPS might be related to mucus modification. Inoculation of either bacterium led to a slow increase in mucus protein content which was however twice as high after E. coli implantation. Inoculation of B. longum in a second step led to a reduction in protein content before B. longum colonized the intestine at a high level suggesting that the protein concentration in the mucus was controlled by the host itself. A new glycoprotein of 200-230 kDa detected during the period preceeding colonization seemed to be broken down by B. longum. The resulting end product might participate in the restoration of E. coli LPS. Finally,B. longum inoculation led to the disappearance of E. coli from kidneys, liver, spleen and lung. The organs were cleared of E. coli before B. longum highly colonized the intestine suggesting that high intestinal colonization by B. longum was not required. Regulation of E. coli invasion seemed to depend on the ability of B. longum to stimulate the immune system.

Bacterial microleakage of Cavit, IRM, and TERM.

In this in vitro study, a model system was developed and tested to evaluate the sealing ability of temporary restorative materials used in endodontic access preparations. The materials studied, Cavit, IRM, and TERM, were tested on 40 premolars against a known bacterial species, Streptococcus sanguis. The leakage of bacterial cells was checked 4 and 8 days after initial immersion in the culture. Thermocycling was introduced on the fourth day. After 8 days the cement thicknesses were measured after the teeth had been longitudinally sectioned. Before and after thermocycling, IRM was less leakproof than Cavit (p < 0.05) and TERM (p < 0.05). Thermocycling aggravated percolation in the case of IRM, and decreased the tightness of Cavit, whereas TERM remained leakproof. The thicknesses were as follows: Cavit, 3.73 mm; IRM, 3.45 mm; and TERM, 5.49 mm. There was no statistically significant relationship between thickness and tightness.

Regulation of the bacterial intestinal implantation in infant born by caesarean section.

Studies were undertaken to determine the regulation of the bacterial intestinal implantation in 19 newborns delivered by caesarean section. Correlation was made with the infant feeding mode. The effect of human milk seemed to be the result of B. bifidum proliferation, in contrast to artificial alimentation that seemed to favour C. perfringens implantation. The question was raised by us as to whether this opposition was only related to alimentation. In fact, B. bifidum itself also had an effect as demonstrated by the lower mean counts of C. perfringens in bottle-fed infants carrying the bifido-bacterial flora (P = 0.05). None of the other faecal bacteria investigated in this study led to the same decrease.

In vitro effects of titanium powder on oral bacteria.

Biomaterial research has been mostly concerned with biocompatibility, i.e. tissue response, of materials for dental or orthopaedic implantation. In this study, the effects of titanium powder on seven bacterial species commonly found in dental plaque or gingival sulcus, were determined by agar incorporation and by liquid medium culture. In neither culture system could any inhibitory or stimulatory activity be detected.

Treatment of diversion colitis by short-chain fatty acids. Prospective and double-blind study.

Diminished production of short-chain fatty acids (SCFA) by altered flora has been suggested in the pathogenesis of diversion colitis (DC). We evaluated prospectively the effectiveness of SCFA irrigation in 13 patients with excluded colon (eight males, five females; mean age, 48 years). The causes of diversion were inflammatory bowel disease (n = 4), colonic cancer (n = 2), sigmoid diverticulitis with perforation (n = 3), ischiorectal abscess (n = 2), and miscellaneous (n = 2). Patients were given, twice a day for 14 days in a double-blind manner, a 60-ml enema containing either SCFA (acetate: 60 mmol/liter; propionate: 30 mmol/liter; and N-butyrate: 40 mmol/liter) (Group 1; n = 7) or isotonic NaCl (Group 2; n = 6). Endoscopy with biopsies was performed before starting the trial (D1) and 14 days later (D14). On D1 all patients had endoscopic and histologic findings suggestive of DC. No endoscopic or histologic changes were observed on D14 in either group. We conclude that endoscopic and histologic lesions of DC were not improved by SCFA irrigation during the 14 days.

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.

Survival of Prevotella intermedia (Bacteroides intermedius) in transport media.

Five transport media were selected for testing, in vitro, the survival of a pure strain of Prevotella intermedia (Bacteroides intermedius) for 6, 24, and 72 hours. Two were non-nutrient transport media (RTF and VMG IV). The three others (TG, PY, PYG) were nutrient media. An increase in the transport time, and manipulations in an aerobic atmosphere compromised the survival of the bacteria. PY ensured good survival of the bacteria. RTF seems to be the best medium for the transport of P. intermedia, when quantitative analysis is required.

[Behavior of Clostridium perfringens in the intestine of newborn infants born by cesarean section]. / Comportement du Clostridium perfringens dans l'intestin du nourrisson né par césarienne.

Colonization of the digestive tract by C. perfringens was studied in infants born by cesarean section. Correlations between the level of colonization and the environment, type of feeding, and presence of other anaerobic bacteria were looked for. Colonization by C. perfringens was found as early as the second day of life in one of the maternity wards studied, suggesting presence of the microorganism in the environment. By the 14th day of life, colonization with C. perfringens was demonstrated in all the bottle-fed or breast and bottle-fed infants. In strictly breast-fed infants, findings suggested antagonism between Bifidobacterium and C. perfringens. Bacteroïdes and Clostridium species other than C. perfringens were not found prior to colonization by C. perfringens.

Bacterial interactions in the intestine of the newborn delivered by cesarean section.

The purpose of this study was to clarify the role of the intestinal anaerobic bacteria colonizing the intestine of the newborn delivered by cesarean section. Control of the intestinal microecology is dependent on many factors including intestinal peristalsis, the intraluminal environment, and microbial interactions, that deter the overgrowth of pathogens populations. Numerous factors help achieve this normal balance. The effect of feeding seems to induce bacteriological changes.

Gram(+) anaerobic intestinal cocci in healthy adults and neonates.

Anaerobic gram(+) cocci were found to be part of the normal indigenous flora of the intestinal tract in healthy adults (19.6%) but they were rarely isolated from healthy neonates (6.1%). A larger variation of species was also present in adults. Their installation in the newborn's intestine seems to be partially inhibited by factors that are not at present very clear.

Nosocomial infections of ocular conjunctiva in newborns delivered by cesarian section.

Colonization of the ocular conjunctiva in newborns delivered by cesarian section occurs usually within the first day of life. We have studied the flora of the ocular conjunctiva at birth, from 19 newborns delivered by cesarian section, coming from two different maternity hospitals. Ocular conjunctiva cultures yielded the main predominant flora in both maternity hospitals considered. The most common genus of this flora are: Staphylococcus, Corynebacterium and Propionibacterium acnes. Peptostreptococcus productus, Neisseria, Eubacterium and Clostridium perfringens are isolated occasionally. In newborns delivered by cesarian section, this flora principally acquired may be the consequence of the presence of bacteria in the ambient air, as well as differences in care provided by the nosocomial personnel.

In vitro survival of Bacteroides intermedius in five transport media, alone or in the presence of Streptococcus sanguis.

The survival rate of Bacteroides intermedius was first tested in monoculture, and Streptococcus sanguis was then added in 5 different transport media; 2 nonnutritious media, the viability-preserving medium of the University of Göteborg No. IV (VMG IV), reduced transport fluid (RTF), and 3 nutritious media, thioglycolate medium (TG), peptone yeast extract medium (PY) and PY medium with 1% glucose (PYG). All manipulations were carried out in an anaerobic chamber. After a given transport time (6 or 24 h) aliquots were spread on plates containing solid PY medium by means of an automated spiral system device, thus permitting counts after incubation. The slight variations in the counts of B. intermedius in monoculture, not exceeding 0.5 log10 in the 5 media tested, indicated its good survival capability, i.e. at least 24 h. By contrast, when S. sanguis was added, it was only possible to use nutritious media such as PY or TG for 6 h. In the glocose-containing media (PYG, TG), the multiplication of rapidly glucose-fermenting microorganisms such as streptococci influenced the B. intermedius survival rate. Therefore, the transport time for oral microbiological samples needs to be reduced as much as possible. The use of excessively rich media (particularly media containing a high level of glucose) should be avoided.

A collaborative study of French laboratories to assess any evolution of antibiotic resistance within the Bacteroides fragilis group.

Antibiotic susceptibility testing of anaerobes by a same methodology allows the authors to draw up suggestions about the evolution of antibiotic resistance within the B. fragilis group. Cefoxitin resistance rates were stable until 1985 and were slowly increasing later. Until 1985 piperacillin was able to inhibit all tested strains. In 1987 the two groups noticed an increasing resistance to piperacillin (4 to 9%). During the 1970's clindamycin resistance was a minor event (less than 1%) then the resistance rate increased rapidly to 10% in 1980. MICs determinations from 1981 to 1985 demonstrated well that clindamycin resistance was stable at this 10% rate. Since 1987 the clindamycin resistance was again increasing and reached respectively 14 to 19% for the two groups of investigators. Metronidazole has kept a good activity against Bacteroides fragilis group strains but some strains with reduced susceptibility (MIC 2 to 8 mg/l) have been described since 1983. Three strains with MIC greater than 8 mg/l were recently described by one of the groups. Until 1987, the clavulanic amoxicillin combination was able to inhibit all strains of the B. fragilis group but only imipenem remained still active on all investigated strains with no change at all for the values of MIC50 and MIC90 determined by the investigators of this study. All these results emphasize the need for periodical surveys within the B. fragilis group in each country.

[Detection of beta-lactamases in the Bacteroides fragilis group]. / Détection des bêta-lactamases des Bacteroïdes du groupe fragilis.

One hundred and thirty three strains of Bacteroides fragilis group isolated from clinical specimen were tested for beta-lactamase activity against five beta-lactam antibiotics, ampicillin, cefaloridin, cefuroxime, cefotaxime and cefoxitin. The Minimal Inhibitory Concentration of antibiotics was determined using the agar dilution method. Beta-lactamase production was detected by a microbiological method in 128 of the 133 (96%) strains. The detected beta-lactamases had a broad-spectrum activity, hydrolyzing both penicillins and cephalosporins (101 strains). Some strains had a wide activity against beta-lactam antibiotics, including cefoxitin (7 strains); among these strains, 3 were found hydrolyzing imipenem.

[Host-Bifidobacterium interactions in the axenic mouse: partial characterization of bifidogenic factors in the intestinal contents]. / Etude des interactions hôte--Bifidobacterium chez la souris axénique: caractérisation partielle des facteurs bifidigènes du contenu intestinal.

Growth factors for Bifidobacterium bifidum were detected in faeces of axenic mice strain C3H. Most of these factors were found in the nondialyzable fraction obtained after aqueous extraction and dialysis. SDS-PAGE and filtration chromatography on Sepharose 4B revealed that many glycosylated components harbored a bifidigenic activity. Intestinal colinization of mice by B. bifidum involved the utilization and eventually the disappearance of the intestinal bifidigenic factors. There was no change in the protein concentration in fecal extracts, but the total hexose concentration was lower. Comparison of electrophoretic PAGE profiles after periodic acid Schiff coloration showed that bacteria used up the glycosylated fractions of many glycopeptides, particularly those of mucins and four glycoproteins. There was no correlation between the hexose concentration detected in every active fraction and the degree of in vivo degradation of bifidigenic factors. The attack on active glycopeptides having a molecular mass greater than 670 kDa thus revealed hexose sites that were not detectable previously by the phenol - sulfuric acid method. However, the amount of bifidigenic factors detected in vitro allowed us to measure the importance of the degradation of a component by B. bifidum in vivo.