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Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups

Arthur C. Ouwehand , Erika Isolauri , Pirkka V. Kirjavainen , Seppo J. Salminen
DOI: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13450.x 61-64 First published online: 1 March 1999

Abstract

The number of bifidobacteria in faeces and intestinal contents has been shown to be reduced with increasing age of the subject. The adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different age. All strains bound significantly less to mucus isolated from elderly subjects, compared to mucus from the other age groups. Two of the tested strains also showed decreased adhesion to mucus isolated from 6-month-old and adult subjects compared to the adhesion to mucus from 2-month-old subjects. The results suggest that reduced adhesion may be a factor involved in the decreasing colonisation of elderly subjects by bifidobacteria.

Keywords
  • Bifidobacterium
  • Adhesion
  • Age effect
  • Mucus

1 Introduction

The composition of the human intestinal microflora has been found to change during the life time of a person. At birth, the gastro-intestinal tract is sterile, but it becomes rapidly colonised by micro-organisms from the birth canal and the environment. The first strains to colonise are facultative anaerobes like Escherichia coli. These organisms are believed to be responsible for the reduction of the redox-potential (Eh) in the intestine. When the Eh is low enough, anaerobic micro-organisms start to colonise the intestine [1]. Among these are the bifidobacteria. They can reach relatively high numbers in the faeces and the intestine of infants due to the presence of N-acetylated sugars (so-called bifidus factor) in breast milk [2] and trans-galactosylated oligo-saccharides in infant formulae [3]. Upon the introduction of solid food and weaning, the numbers of bifidobacteria decrease and stay at a more constant level throughout adult life. At old age, however, the number of bifidobacteria has been found to decrease [4, 5].

Adhesion to the intestinal mucosa is regarded a prerequisite for colonisation [6] and is one of the selection criteria for probiotic micro-organisms [7]. We have previously shown that some probiotic strains adhere well to enterocyte like Caco-2 tissue culture cells [8] and intestinal mucus [9]. Adhesion of the probiotic strains to intestinal mucus has been found to be slightly, but significantly, influenced by age [9]. We hypothesised that one factor causing the reduction in the number of bifidobacteria in the intestinal microflora of the elderly may be reduced adhesion to the intestinal mucosa. Therefore, the adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different ages: newborns, 2- and 6-month-old infants, adults and elderly subjects.

2 Materials and methods

2.1 Bacteria and culture conditions

Four Bifidobacterium strains were used; Bifidobacterium lactis Bb12 (Chr. Hansen, Denmark), Bifidobacterium 420 (Visby, Denmark), Bifidobacterium 913 (Visby, Denmark) and Bifidobacterium BF1100 (Visby, Denmark). The strains were a generous gift from Dr. M. Saxelin (Valio, Helsinki, Finland). The bacteria were grown in 5 ml Reinforced Clostridial Medium (Oxoid) from stocks stored at −75°C in 40% glycerol (1% inocculum). To the medium, 50 µl of tritiated thymidine ([methyl-1,2-3H]thymidine, 117 Ci mmol−1) was added to metabolically radiolabel the bacteria. After overnight growth at 37°C, the bacteria were harvested by centrifugation (2000×g) and washed twice with phosphate buffered saline (PBS; pH 7.2; 10 mM phosphate) and resuspended in PBS. The absorbance at 600 nm was adjusted to 0.5±0.02 in order to standardise the number of bacteria (107–108 CFU ml−1).

2.2 Mucus preparation

Mucus was prepared from human faecal samples essentially as described previously [9]. The faecal samples were obtained from healthy newborns (n= 28), 2- and 6-month-old infants (n= 11 and 17, respectively), adults (n= 14; age 25–52 years) and elderly subjects (n= 30; age 74–93 years). Equal amounts of mucus from individuals of each age group were pooled. The pooled mucus was dissolved in HEPES (N-[2-hydroxyethyl]piperazine-N′-2-[ethane sulfonic acid])-Hanks buffer (HH; 10 mM HEPES; pH 7.4) at a concentration of 1 mg ml−1. Any particulate material was removed from the suspension by centrifugation (2000×g for 10 min).

2.3 Mucus characterisation

The amount of mucin was determined according to the method described by Hall et al. [10] using bovine sub-maxillary mucin as standard. Carbohydrate was determined as described Dubois et al. [11] with glucose as standard. Protein was determined with a modified method of Lowry et al. [12] as described by Miller and Hoskins [13], with bovine serum albumin as standard. The results are shown in Table 1.

View this table:
Table 1

The properties of intestinal mucus isolated from subjects of different age

Mucus sourceCarbohydrate (%)Protein (%)Mucin (%)
Newborn29.130.066.9
2-Month-old infants15.349.471.6
6-Month-old infants31.337.273.1
Adults (25–52 years)17.130.472.2
Elderly (74–93 years)40.113.363.8
  • In percent by weight.

2.4 In vitro adhesion assay

The adhesion of the radioactively labelled bacteria was determined as described previously [9]. The adhesion was expressed as the percentage of radioactivity recovered after adhesion, relative to the radioactivity in the bacterial suspension added to the immobilised mucus.

2.5 Statistical analysis

The results from the adhesion experiments are expressed as the average of four to six independent experiments. Each experiment was performed with three parallels. A two-tailed t-test was used to evaluate the statistical significance (P<0.05) of the differences in the ability of each bacterial strain to adhere to two different mucus preparations and between the adhesion of two strains to mucus from the same origin.

3 Results

The adhesion of the four Bifidobacterium strains to mucus is shown in Fig. 1. The adhesion of Bifidobacterium BF 1100 to mucus from adult subjects was significantly less (P<0.05) than the adhesion of B. lactis Bb12 to the same mucus; 4.2% (S.E. 1.9) and 11.3% (S.E. 2.4), respectively. No other significant differences were observed in the adhesion between the strains tested (P > 0.05). All strains adhered significantly less (P<0.05) to mucus isolated from elderly subjects (0.1–1.6%) than to mucus isolated from subjects of younger age (4.2–12.3%). Adhesion of Bifidobacterium 913 to mucus from 2-month-old subjects was significantly higher (P<0.05) than mucus from 6-month-old and adult subjects; 11.5% (S.E. 3.8) vs. 8.6% (S.E. 3.3) and 5.9% (S.E. 2.0), respectively. Also, the adhesion of Bifidobacterium BF1100 was significantly higher (P<0.05) to mucus from 2-month-old subjects than to mucus from adults; 8.1% (S.E. 3.4) and 4.2% (1.9%), respectively.

Figure 1

The adhesion of four Bifidobacterium strains to intestinal mucus isolated from subjects of different age: newborn, 2- and 6-month-old infants, adults and elderly. Adhesion is expressed as the percentage of bacteria adhered to immobilised mucus compared to the amount of bacteria added to the mucus. a, Adhesion to mucus from elderly subjects is significantly different from adhesion to all other types of mucus. Bars sharing the same characters, b, c, d and e, show significantly (P<0.05) different adhesion. Results are shown as the average of four to six experiments, error bars indicate the standard error.

4 Discussion

Adhesion to the intestinal mucosa with consequent colonisation of some areas of the gastrointestinal tract, is considered a factor important for colonisation of the micro-flora [6, 7]. In the present study, the adhesion of two of the four tested strains to mucus isolated from adult subjects was found to be significantly different. In all other cases, no significant difference was observed in the adhesion between the tested strains to the same mucus type. All four strains were found to bind significantly less to mucus from elderly subjects when compared to mucus from all other age groups (though elderly subjects are, of course, also adults, they are regarded here as a separate group). Two strains also showed reduced adhesion compared to mucus isolated from 2-month-old subjects; Bifidobacterium 913 adhered less to mucus from 6-month-old infants and adults, and Bifidobacterium BF 1100 adhered less to mucus from adults. We previously showed that the adhesion of several probiotic bacteria was significantly higher to mucus isolated from adult subjects as compared to mucus isolated from infants [9]. The adhesion of B. lactis Bb12 was, however, not significantly different for the tested age groups (newborns, 2- and 6-month-old infants and adults). A similar observation was made in the present study for those age groups.

In an early study by Mitsuoka and Hayakawa [4], it was suggested that the number of bifidobacteria in the faecal flora was significantly reduced in elderly subjects compared to younger adults. The results presented in this study suggest that reduced binding to intestinal mucus may explain some of the observed reduction in the number of bifidobacteria in elderly subjects.

The mucus preparation of the elderly subjects differs considerably from those of the other preparations; it contains more carbohydrate and less protein. The total amount of mucus is, however, similar. For most bacteria, interaction with the carbohydrate moieties of the mucin molecule is important for binding. Sato and co-workers [14] have shown that bifidobacteria bind to polysaccharides. It is not clear if the reduced adhesion is due to differences in glycosylation or degradation of the mucus, since the mucus was not characterised into such detail. In either case, adhesion is likely to be affected, possibly also in vivo, with an impact on the intestinal colonisation and an alteration of the microbial balance both on the mucosal surfaces and in the intestinal lumen.

Bifidobacteria have been suggested to function as an anti-infectious barrier to pathogenic bacteria [15]. Increasing their numbers may, therefore, be desirable. The results presented here suggest, however, that when considering administering probiotic bifidobacteria to elderly subjects, special strains should be selected.

Acknowledgements

We wish to thank the staff from the Mäntyrinne and Luolavuori nursing homes for collecting the faecal samples from the elderly subjects and Pia Niemi for technical assistance.

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