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Collagen (Cn-I) binding by gut lactobacilli
Abstract
Four gut Lactobacillus strains displaying the features which make them particularly promising for the preparation of probiotic products were investigated together with 5 fresh isolates and one collection strain of Lactobacillus plantarum for their ability to bind type I collagen (Cn-I). Immobilised Cn-I in microtitre plates was bound only by 3 strains of gut lactobacilli from piglets and the collection strain Lactobacillus plantarum LHI 10 from Prague in range of A570nm readings 0.114-0.221. Six strains (isolates from turkeys and a calf) did not bind Cn-I (A570nm < 0.1) in this assay. An influence of cultivation medium on Cn-I binding was significant (P < 0.001) in all four adherent strains. Significantly higher (P < 0.001) binding of Cn-I was observed for Lactobacillus casei L 81 and Lactobacillus plantarum LHI 10 grown on solid medium (MRS agar) than for MRS broth-grown cells, however, Lactobacillus plantarum L 5 and Lactobacillus fermentum L 435 expressed significantly (P < 0.001) higher Cn-I binding during cultivation in MRS broth. The specificity of the binding was confirmed because the Cn-I binding by lactobacilli after their preincubation with this protein was completely abolished. Three selected inhibitors (fucoidan, heparan sulphate and hyaluronic acid) significantly (P < 0.001) reduced Cn-I binding by the Lactobacillus plantarum L 5 strain. Following up on some earlier strain characteristics, these results suggest that the selected piglets lactobacilli are also able to bind Cn-I and therefore should antagonize collagen niche colonization by various enteropathogens when used for probiotic purposes.
... Lactobacilli were classified as strongly adherent (A 55 onm > 0.3), weakly adherent (0.1 < A550nm > 0.3), or nonadherent (A550nm < 0.1) (Styriak et al., 1999a). ...
... Blank wells were used as negative control. Lactobacilli were classified as strongly adherent (Assonm > 0.3), weakly adherent (0.1 < Assonm > 0.3), or nonadherent (Assonm < 0.1) (Styriak et al., 1999a). The specificity of binding was tested by !-hour preincubation of bacteria with an equal volume of type-1 collagen solution at concentration of I 00 ~g mr 1 and subsequent washing followed by examination of bacterial binding to the same protein. ...
... Aleljung (1991) demonstrated that binding to solubilized collagen is frequently expressed among Lactobacillus strains of different origins; 75% of their LAB isolates bound solubilized type I collagen. Adherence of vaginal (Styriak et al., 2001) and intestinal lactobacilli (Styriak et al., 1999a) of porcine and bovine origin to collagen-I has also been demonstrated. In the current study only 3 porcine lactobacilli strains, from the total 13 were able to adhere to Collagen-! with SHVD FC71 expressing the greatest binding potential. ...
... Some isolates have been shown to specifically bind collagen, one of the extracellular matrix (ECM) proteins primarily found in connective tissue [1,2,37,39]. The extracellular matrix is a mixture of secreted proteins composed primarily of collagens, fibronectin, laminin, and proteoglycans located on epithelial and endothelial cell surfaces. ...
... Although several studies have demonstrated collagen binding [1,2,37,39], we have shown fibronectin binding by lactobacilli. Earlier studies by other workers have suggested that fibronectin binding may mediate adherence to epithelial cells by lactobacilli [8,38,39]. ...
Lactobacilli are members of the normal mucosal microflora of most animals. Many isolates of Lactobacillus spp. are adherent to epithelial cells. In this study, using Lactobacillus acidophilus and L. agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins. Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected. In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers. Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin. Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces.
... Crude intestinal mucin was prepared from the small intestine of a weaned pig according to the method described by Š t y r i a k et al. [36]. were then overlaid with 3 ml of 0.7 % PYG agar, which was seeded with 0.3 ml overnight culture of pathogen. ...
The effects of zinc sulphate on selected properties of L. plantarum CCM 7102 were tested in vitro . The resistance of lactobacilli to higher concentrations of ZnSO 4 (up to 5000 mg Zn ²⁺ .l ⁻¹ ) in growth media was strain-dependent. Further studies were carried out on the most resistant strain of L. plantarum CCM 7102. While the addition of low concentrations of zinc sulphate into the growth media (< 100 mg Zn ²⁺ .l ⁻¹ ) did not influence the properties of L. plantarum CCM 7102, the concentrations of 100—500 mg Zn ²⁺ .l ⁻¹ stimulated: the growth rate, production of lactic acid, adhesion to porcine enterocytes and the inhibition of pathogens E. coli O8:K88 ⁺ ent ⁺ , S. enterica and S. Typhimurium . Conversely, however, high concentrations > 500 mg Zn ²⁺ .l ⁻¹ inhibited these properties. The addition of zinc (250 mg Zn ²⁺ .l ⁻¹ ) did not affect the resistance to antimicrobials, low pH, and the resistance to bile salt was affected only weakly. Zinc-resistant probiotic Lactobacillus strains are suitable for use in feedstuffs with a higher content of zinc designed for the prevention of post weaning diarrhoea in pigs.
... In case of damaged mucosa, ECMs can be exposed allowing undesirable microbial colonization and infection (Styriak et al., 2003). However, because of the ability of some lactobacilli to adhere to these matrices, they can compete with pathogens for the same receptors and occupy their potential binding sites in the gut (Styriak et al., 1999;Neeser et al., 2000;Lorca et al., 2002). One of the reported ECM adhesins is the collagen-binding protein (CnBP) of L. reuteri NCIB 11951 (Table 1). ...
Despite the increasing number of scientific reports describing adhesion of Lactobacillus to components of the human intestinal mucosa, information on the surface molecules mediating this adhesion and their corresponding receptors is fragmentary. This MiniReview compiles present knowledge of the genetically and functionally characterized Lactobacillus factors responsible for mediating adhesion to different components of the human gastrointestinal tract. In addition, for the proteins among these factors, the domain structure is discussed, and where appropriate the results of in silico analyses are reported.
... Moreover, ECM binding ability has been shown to be expressed by several pathogenic bacteria and to promote bacterial virulence (Lowrance et al. 1990;Patti et al. 1994;Hienz et al. 1996). Selected probiotic bacterial strains should be able to compete with pathogens for the same receptors and to occupy their potential binding sites in the gut (Neeser et al. 2000) including collagen-I (Š tyriak et al. 1999a;Lorca et al. 2002) and fibronectin (Lorca et al. 2002). ...
The aim of this study was to investigate extracellular matrix (ECM) and mucin binding of selected bacterial isolates with probiotic features in comparison with commercially used probiotic bacteria.
ECM molecules were immobilized in microtitre plates (mucin and fetuin) or on the surface of latex beads. Porcine mucin was bound by all 13 probiotic strains tested with important inter-strain differences; however, fetuin binding was similar (weak) for all 14 strains tested. Strongly positive (three) binding of bovine fibrinogen was expressed by strains from fermented food (Lactobacillus rhamnosus GG, L. casei Shirota and L. johnsonii La1) as well as by L. casei L.c., Lactobacillus sp. 2I3 and by L. plantarum LP. The other strains expressed moderate (2) or weakly positive (1) binding of bovine fibrinogen. Strongly positive (3) binding of porcine fibronectin was observed only with two strains; however, all other strains also bound this molecule. Bovine lactoferrin was bound to a higher extent than transferrins.
Some animal strains (at least L. casei L.c. and Lactobacillus sp. 2I3) are comparable with the commercially used strains with respect to their ECM binding ability. As this feature is important for probiotic bacteria to be able to colonize intestine, these strains should be considered for their wider use in fermented feed (or probiotic preparations) for animals.
... Microscopic evaluation can be used after fixation and Gram staining of the bacteria (Tuomola and Salminen, 1998), but the method is equally laborious as plating. When the amount of released bacteria is sufficient for turbidometric analysis, spectrophotometry can be used (S ˇ tyriak et al., 1999), but the sensitivity and accuracy are maybe less compared to plating or microscopic enumeration. These methods cannot be used when adhesion of one bacterial strain is studied in an environment where other bacteria are present. ...
The adhesion of bacteria to host tissue is the first step in pathogenesis. Similarly, bacterial adhesion to inanimate surfaces is the first step in formation of biofilms-a real problem in industrial processes and medical devices. Various agents capable of blocking the adhesion of bacteria to surfaces have been identified, such as probiotics, which are supposed to prevent the adhesion of pathogenic bacteria to the intestinal mucosa. Although measurement of bacterial adhesion is important itself, especially when agents used to prevent adhesion are developed, a relative small number of techniques can be used in the measurement of adhesion. These techniques are not well validated and there is lack of studies where those methods are compared to each other. Here we have compared different commonly used methods to measure adhesion of bacteria; radioactive labelling, fluorescence tagging, and staining of bacteria. The methods were used to measure the adhesion of Escherichia coli and Salmonella enterica serovar Typhimurium to intestinal mucus. Moreover, selected probiotic strains were used to study whether probiotics or the adhesion method used affected the results. As a result, we show that the best reproducibility and sensitivity were obtained using radioactive labelling. With other methods, the sensitivity was too low due to poorly adhering bacteria and low signal-to-background ratio.
Ten auto aggregating vaginal Lactobacillus strains (five of these strains were selected among isolates from sows' vaginal swabs and the other five among isolates from cows' vaginal swabs) were investigated for their ability to bind type I collagen (Cn-I). All 10 autoaggregating strains in the range of A570nm readings 0.118-1.806 bound to immobilised Cn-I (at concentration of 100 μg/ml) in wells of microtitre plates, however, Lactobacillus acidophilus SV31 was much more adherent than the rest of the tested strains. The influence of culture medium on Cn-I binding was confirmed only in 50% of the tested strains when agar-grown cells bound significantly more Cn-I than broth-grown cells. The specificity of the binding was confirmed since the Cn-I binding by lactobacilli was abolished after their preincubation with this protein. The effect of heparan sulphate and hyaluronic acid was tested on 5 vaginal strains displaying the best Cn-I binding in microtitre plates after their cultivation on MRS agar plates. Both selected inhibitors significantly (P < 0.001 or P < 0.01) reduced Cn-I binding by the majority of strains. The presence of the gene coding APF (aggregation-promoting factor) was detected in seven strains (all five sows' and two cows' Lactobacillus strains) by PCR.
Urogenital tract infections affect a very high number of women worldwide, producing many clinical situations that imply increasing costs to the health systems, and a consequent morbility and mortality. The Urinary Tract Infections are more common in pre pubers or postmenopausal women, while the Genital Infections are more related to Sexually Active Women. Many of the applied therapeutics imply the use of antibiotics or other drugs that produce adverse effects not only in the urogenital tract, but at general level. This tract is also affected by other external or internal factors that have effect on the equilibrium of the urogenital microbiota, increasing the incidence of infections. During the last years the preventive measures are being applied around the world. In the urogenital tract, they are considerably important by the relationship with pregnancy development, newborn status and mother complications. The application of probiotics for many clinical situations, and for the restoration of the urogenital microbiota and prevention of infections, is more and more frequent. Probiotics are defined as live microorganisms administered in high numbers to the host to produce a physiological effect. The mechanisms involved in the probiotic effect include the production of antagonistic substances, the competitive exclusion phenomenon, the competition for nutrients, the colonization ability, the biofilm formation and/or the stimulation of the immune system. Some of these mechanisms have been showed by "in vitro" assays, but not yet in many "in vivo" experiments. The diversity of strains claimed as probiotics is higher every time, without the publication of clinical trails supporting their beneficial effect. These aspects and other related with the rationale of probiotic application in the urogenital tract are discussed in the present review.
Adhesion to the intestinal mucosa is one of the main selection criteria for probiotics. Selection of adhesive strains is performed in vitro. Little is known, however, as to how well in vitro adhesion correlates with in vivo adhesion; to date, this has not been systematically reviewed. Because the various adhesion models usually describe only one part of the intestinal mucosa, a combination of models may provide the best information. Incubation conditions affect the outcome of an adhesion assay and should be optimized for physiological relevance. The effects of digestion, the food matrix and the normal microbiota appear to have a significant influence, but have not been extensively investigated. Many factors interfere with the mucosal adhesion of probiotics, it is therefore difficult to extrapolate in vitro adhesion results reliably to the in vivo situation in humans. It is suggested that the relationship between in vitro and in vivo adhesion and the significance of adhesion for probiotic efficacy should be further investigated. In determining the in vivo importance of adhesion and the assumed relationship between in vitro and in vivo adhesion, adhesive and low-adhesive isogenic strains would appear to be the best tools.
Ten gut and ten vaginal Lactobacillus strains were investigated for their ability to bind type I collagen (Cn-I) and four selected gut lactobacilli were investigated for their binding to other extracellular matrix (ECM) molecules. Immobilized Cn-I (100 mg/L) in wells of microtitre plates was bound by all 10 autoaggregating vaginal strains and by 3 strains of gut lactobacilli from piglets in the range of A570 readings 0.114-1.806. L. acidophilus strain SV31 was much more adherent than the rest of strains. All four gut lactobacilli tested for binding to other ECM molecules displayed good binding to porcine fibronectin and heparin and some of them bound weakly to fetuin and porcine mucin. No binding of these strains was observed to bovine mucin, bovine fibrinogen and bovine lactoferrin.
The ability of Lactobacillus casei strain KE99 to reduce sulfide, ammonia, and to adhere to bio-surfaces was characterized and compared with three lactobacillus reference strains. Sulfide reduction by strain KE99 in MRS broth increased exponentially after 10-h growth and reached a maximum (>300 ppm reduction) within 48 h. KE99 demonstrated a maximum reduction of sulfide under anaerobic (341 ppm) growth conditions at pH 6.0-8.0 range. Maximum anaerobic reduction of sulfide was demonstrated by L. casei 393 at pH 7.0 (272 ppm); L. rhamnosus at pH 8.0 (277 ppm); and L. reuteri at pH 7.0 (244 ppm). KE99 reduced sulfide more (p < 0.0001) in MRS broth spiked with Na2S (374 ppm) than (NH4)2S (340 ppm) salts. Ammonia reduction by strain KE99 and the three lactobacillus reference strains in MRS broth was low. Ammonia reduction reached a maximum within 36 h and remained unchanged over extended incubations of 48 h to 72 h or further. KE99 reduced ammonium sulfate (37 ppm) more readily than the nitrate (31 ppm), hypophosphate (29 ppm), or chloride (20 ppm) salts of ammonia. KE99 and the three reference strains of lactobacilli demonstrated avid binding to Bio-coat (Cn type-I, Cn type-IV, laminin, fibronectin), Matrigel, and Caco-2 cell monolayers in vitro. The number of lactobacilli binding to Caco-2 was estimated at 74/cell with strain KE99, which was significantly higher compared with 40/cell (p < 0.0001), 26/cell (0.0001), and 64/cell (p < 0.002) with L. casei 393, L. reuteri, and L. rhamnosus, respectively. The interaction of KE99 to immobilized Cn type-I was saturable and reached an equilibrium within 1 h at room temperature. KE99 binding to Cn type-I occurred at a wide pH range and was biphasic with maximum binding at pH 5.5 and 7.5. Inhibition and binding-displacement experiments with different salts and sugars suggested that the KE99 binding to immobilized Cn type-I may involve a combination of electrostatic and lectin-type interactions. KE99 effectively detached the Cn-adherent E. coli O157:H7 in the range of 55% (ATCC43895) to 76% (ATCC43894). The binding-displacement values for L. casei 393, L. reuteri and L. rhamnosus to detach Cn-adherent E. coli O157:H7 (ATCC43894) were 66 +/- 4%, 59 +/- 2%, and 64 +/- 2%, respectively. Also, a reconstituted solution of the freeze-dried KE99 preparation effectively detached the Cn-adherent E. coli O157:H7 in a dose-dependent manner that reached a binding-displacement equilibrium of 85% at a 1% wt/vol KE99 concentration.
An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.
Three gut lactobacilli from piglets (Lactobacillus plantarum L 5, Lactobacillus paracasei L 81, Lactobacillus fermentum L 670) and Lactobacillus casei subsp. pseudoplantarum L.c.) from a calf were examined by microtitre plate binding assay for their lectin-like binding activity after their cultivation on Rogosa agar and in MRS broth. Three ECM (extracellular matrix) molecules (fetuin, porcine fibronectin and porcine mucin) were selected for this assay. Additionally, the effect of heparin on the binding of these three ECM molecules by Lactobacillus strains in microtitre plates was tested. Moreover, haemagglutination tests with pig, cattle, sheep, and hen erythrocytes were performed. However, none of the four Lactobacillus strains examined did react with any of the erythrocytes tested. The differences between individual strains were observed in their binding to immobilised ECM molecules. The best adherent was the Lactobacillus plantarum L5, however, the other three strains showed also good ECM binding. With regard to an influence of cultivation medium on lectin-like binding activity, binding of all ECM molecules was expressed in Lactobacillus paracasei L 81 to significantly higher degree (P < 0.001) after cultivation on Rogosa agar than in MRS broth. Similarly, strains Lactobacillus fermentum L 670 and Lactobacillus casei subsp. pseudoplantarum L.c. displayed significantly higher (P < 0.001) binding of fibronectin and mucin after growth on Rogosa agar in comparison with MRS broth cultivation. However, no significant (on fetuin and fibronectin binding) or opposite effect (on mucin binding) of cultivation medium was observed in Lactobacillus plantarum L 5 strain. The influence of cultivation medium on fetuin binding by Lactobacillus fermentum L 670 was also not significant while Lactobacillus casei subsp. pseudoplantarum L.c. bound fetuin significantly better (P < 0.01) after growth on Rogosa agar. Heparin pretreatment increased the binding of the ECM molecules by the Lactobacillus fermentum L 670 strain significantly (P < 0.001 or P < 0.05) with the exception of porcine fibronectin when the strain was cultivated in MRS broth. This result is important especially in the connection with the previous observations that heparin decreased ECM binding of enteropathogens as staphylococci or clinical enterococcal isolates. Following up on some earlier strain characteristics, these results indicate that the selected lactobacilli are probably suitable for probiotic purposes.
The effect of application of omega-3 polyunsaturated fatty acids (omega-3 PUFA) on intestinal colonization by Lactobacillus paracasei and on cellular immunity has been investigated in gnotobiotic pigs. The administration of polyunsaturated fatty acids positively affected the adhesion of Lactobacillus paracasei to the jejunal mucosa of gnotobiotic piglets. When compared to the control group, the number of Lactobacillus paracasei adhering to the jejunal mucosa was by 12% higher in piglets of the experimental group (5.10 log 10/cm2 vs. 4.55 log 10/cm2). The respective counts of Lactobacillus paracasei adhering to the ileal and colonic mucosa of 28 day old gnotobiotic piglets reached 4.45 and 5.05 log 10/cm2 in group C and 4.44 and 4.95 log 10/cm2 in group E. Omega-3 PUFA supplementation increased the phagocytic activity of neutrophils by almost 100% on day 28 of life as well as the subpopulations of lymphocytes (CD8) in the peripheral blood of germ-free piglets on day 21 of life. Our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA. The stimulatory effect of PUFA upon adhesion of lactobacilli could be used for enhancing the effectiveness of probiotics in inhibiting digestive tract pathogens.
The effect of the inoculation of three Lactobacillus plantarum strains upon lactic, acetic, acetoacetic and propionic acid levels in the mucosal film (F) and the jejunal and ileal contents (O) has been investigated in gnotobiotic pigs. In the jejunum of the inoculated animals, the mucosal film revealed significantly increased levels of lactic, propionic and acetoacetic acids when compared to the contents (25.3 vs. 10.8 mmol.l-1, 18.5 vs. 5 mmol.l-1 and 29.7 vs. 11.2 mmol.l-1, respectively) as well as insignificantly increased acetic acid levels (11.0 vs. 5.8 mmol.l-1). In the ileum of gnotobiotic pigs, propionic acid levels of the mucosal film were significantly higher than those of the contents (21.2 vs. 9.5 mmol.l-1, p < 0.05). In comparison to the contents, the increased lactic, acetic and acetoacetic acid levels in the film proved to be insignificant. The above results suggest that the significantly increased levels of the Lactobacilli-produced organic acids may present an efficient barrier inhibiting the adherence of digestive tract pathogens to the intestinal mucosa.
125I-labeled type I collagen (Cn-I) binding of 92 fresh isolates and 18 type culture collection strains of lactobacilli was tested. More than 75% of the strains bound Cn-I. The binding was inhibited by excess of unlabeled Cn-I, gelatin, and was sensitive to proteinase K. Other proteins such as fibronectin and albumin and various carbohydrates such asD-galactose,D-fucose, andD-mannose did not inhibit the binding. Therefore, we propose binding of Cn-I to lactobacilli involving specific surface protein(s).
Strains of enteropathogenic Escherichia coli (EPEC, 157 strains), enterotoxigenic E. coli (ETEC, 10 strains), enteroinvasive E. coli (EIEC, 40 strains), enterohaemorrhagic E. coli (EHEC, 10 strains), and enteroadherent E. coli (EAEC, 6 strains), all isolated from children and adults with diarrhoea, uropathogenic E. coli (25 strains) and faecal E. coli (36 strains) isolated from healthy persons were tested for binding to subepithelial connective tissue proteins, viz. fibronectin, collagen and vitronectin (S-protein). Strains expressing high and moderate binding to these proteins were found in all groups including normal stools. The highest incidence of binding strains were found among EAEC, EHEC and EPEC strains. Many strains bound collagen only whereas no strain bound vitronectin only. Binding to these proteins was generally best expressed after overnight growth on CFA agar at 37 degrees C. It is not correlated to surface hydrophobicity, and it is not influenced by O antigens or K1 and K5 antigens. The presence of fimbrial adhesins on extraintestinal isolates did not enhance the binding to soluble form of the matrix proteins. During pathological conditions when subepithelial connective tissue proteins are exposed, strains with the ability to bind fibronectin, collagen and/or vitronectin may have a selective advantage to colonize the tissue.
This chapter discusses the binding of extracellular matrix (ECM) proteins by microbes. Proteins expose different microbial-binding domains when they are in soluble form and when they are immobilized in the ECM or on a biomaterial surface. This is confirmed by the finding that Yad A of Y. enterocolitica and P fimbriae of uropathogenic E. coli bind surface immobilized fibronectin (Fn) but not serum Fn. Binding of only immobilized or soluble forms of ECM proteins suggests that binding occurs to an epitope which is sensitive to conformational changes of the ECM molecules. Several of the identified ECM-binding structures on Staphylococcus aureus (S. aureus) and other bacteria are proteins, and hence the use of heat-killed bacteria may render false low or negative results. Proteases are produced by many microbial pathogens, and fibronectinolytic activity has been detected in strains of S. aureus, Staphylococcus epidermidis, Propionibacterium acnes, and a number of other anaerobic bacterial species. Binding of Fn by proteolytic bacterial strains may thus permit subsequent analysis of binding domains if the Fn is analyzed after proteolytic degradation in parallel. Most bacteria seem to express binding of ECM proteins during the exponential growth phase. The chapter also discusses ECM constituents, binding of soluble proteins and glycosaminoglycans, binding of immobilized proteins, binding of ECM proteins immobilized in tissue in vitro, and characterization of binding of ECM.
In the present study, the effect of Lactobacillus casei subsp. casei and Lactobacillus fermentum inoculation on jejunum and ileum colonization in gnotobiotic piglets has been observed. The characteristic features of the strains used were strong adherence to pig epithelial gut cells as well as inhibitory activity against enteropathogenic E. coli under in vitro conditions. Strains were inoculated to 2, 3, and 4 day old gnotobiotic piglets at a dose of 2 ml (1 x 10(8) germs/ml). On the second day after the last inoculation, Lactobacillus casei subsp. casei strain counts adhered to the jejunum mucosa and those adhered to the ileum mucosa were 4.54 log 10.cm-2 and 5.40 log 10.cm-2, respectively. Lactobacillus fermentum counts adhered to the jejunum mucosa and those adhered to the ileum mucosa were 5.73 log 10.cm-2 and 4.01 log 10.cm-2, respectively. On day 5 after the last inoculation, the counts in both strains were by one log higher. The results obtained point out to the fact that Lactobacillus casei subsp. casei and Lactobacillus fermentum colonized the mucosa of both jejunum and ileum and survived in the intestinal tract. The adherence of lactobacilli to gut cells in vitro correlated with their capability to adhere to the mucosa of both jejunum and ileum in vivo.
The effect of inoculation of Lactobacillus casei on selected parameters of metabolic profile and intestinal metabolism of gnotobiotic piglets was investigated during the first three weeks of their life. The experiment was carried out on 8 germ-free piglets. The experimental group was inoculated once a day with the Lactobacillus casei subsp. casei strain. The inoculum contained 1 x 10(8) microorganisms in 1 ml. The control group of piglets received no inoculum. Lactobacillus casei colonized jejunum and ileum in the numbers from 5.63 to 6.06 log 10 cm-2 and their numbers in the jejunal and ileal contents were in the range 8.38-9.87 log 10.ml-1. The daily consumption of milk by the inoculated animals was significantly higher (p < 0.001). The average weight of inoculated piglets at the end of the period investigated was higher by 29.7%. Lactobacillus casei affected several parameters investigated. Piglets inoculated with lactobacilli showed significantly lower (p < 0.05-0.01) values of pH of the jejunal content, numbers of erythrocytes, values of haematocrit, urea, glucose, total lipids, cholesterol and calcium in the serum and significantly higher values (p < 0.05-0.01) of lactic acid in the jejunal content. The values of phagocytic activity and the index of phagocytic activity in the piglets of the experimental group were two to three-fold higher in comparison with those detected in the control group. The application of Lactobacillus casei affected positively the growth of gnotobiotic piglets, their intestinal metabolism, the level of cholesterol in the serum and phagocytic activity.