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Letters in Applied Microbiology 2005, 40, 391–396

doi:10.1111/j.1472-765X.2005.01684.x

Adherence and colonization properties of Lactobacillus rhamnosus TB1, a broiler chicken isolate T. Bouzaine1, R.D. Dauphin2, Ph. Thonart2, M.C. Urdaci3 and M. Hamdi1 1

UR-Microbial and Food Process, National Institute of Applied Science and Technology, University of Carthage, Tunis, Tunisia, Center Wallon of Industrial Biology, Faculty of Agronomy, Gembloux, Belgium, and 3Laboratoire de Microbiologie, ENITA-University of Bordeaux, Gradignan, France

2

2004/0716: received 22 June 2004, revised 30 January 2005 and accepted 31 January 2005

ABSTRACT T . B O U Z A I N E , R . D . D A U P H I N , P H . T H O N A R T , M . C . U R D A C I A N D M . H A M D I . 2005.

Aims: Selected lactic acid bacteria (LAB) isolated from intestinal tract of chicken have been studied in order to investigate their ability to adhere in vitro to Basement Membrane Matrigel (BMM). A selected strain showing a good adherence in BMM test was used for in vivo colonization assays. Methods and Results: In vitro assessment of adhesion of broiler chicken isolates was performed using BMM assay. Among LAB strains tested, Lactobacillus rhamnosus TB1 showed a good adherence that was comparable to the one of an Escherichia coli EPEC strain used as positive control. For in vivo colonization assays this strain was fluorescently stained with the carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) thus allowing its detection in different layers of intestinal tract after inoculation in broiler chicken. Further, stained L. rhamnosus were found with a highest value in rectum, jejunum and ileum both 3 and 24 h after administration. Conclusions: BMM assay is a quick method to test in vitro adhesion properties of bacterial strains and cFDASE-stained bacteria may be considered as an alternative method to test in vivo adhesion and colonization properties. Significance and Impact of the Study: Lactobacillus rhamnosus TB1 was therefore showed to be able to adhere strongly in vitro to BMM and in vivo to intestinal epithelial cells of chicken and may be considered as a potential probiotic for chicken. Keywords: adhesion, cFDA-stained bacteria, chicken, gastrointestinal tract, Lactobacillus rhamnosus, Matrigel.

INTRODUCTION Gastrointestinal microbiota is considered as the first biological barrier against pathogenic bacteria. Probiotic microorganisms have been scientifically shown to influence the composition and activity of the intestinal microbiota and have beneficial effects in the host by preventing or reducing the duration of some gastrointestinal infections (Salminen et al. 1996; Ouwehand et al. 2002). It has been suggested that the possible action mechanisms of probiotics involve a Correspondence to: Moktar Hamdi, Microbial and Food Process, INSAT, University of Carthage, Boulevard de la Terre BP. 676, 1080 Tunis, Tunisia (e-mail: [email protected]).

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pH reduction of the gut, direct antagonism by production of antimicrobial compounds, competition for the adhesion sites and available nutrients and stimulation of the immune system (Collins and Gibson 1999). Knowledge about the importance of adhesion as a colonization factor is lacking, although adhesiveness is considered as a criterium to choose a probiotic strain (Reid 1999). In addition, adherence of probiotics to intestinal epithelial cells and the ensuing temporary colonization of the gut are probably of crucial importance for their beneficial health effect (Bernet et al. 1994; Hudault et al. 1997). Moreover, adhesion is also considered important in immunomodulation properties of lactic acid bacteria (LAB) (Blum et al. 2002). The difficulties in assessing adherence of

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probiotic strains in vivo have led to the development of in vitro adherence assays (Blum et al. 1999). Various tissue targets like tissue pieces, isolated epithelial cells, cultured cells as Caco-2 and HT-29, isolated extracellular matrix (ECM) proteins, and the intestinal mucus, have been proposed to test the adhesion of LAB. The level of adhesion has been enumerated either by microscopic visualization, bacterial culture counts or by using metabolically radiolabelled bacteria (Horie et al. 2002). ECM is a relatively stable structure that underlies and surrounds connective tissue cells. Matrigel is a reconstituted basement membrane preparation used in bacterial adhesion and invasion studies (Kukkonen et al. 1993; Horie et al. 2002). Selected probiotic strains should be able to compete with pathogens for the same receptors and to occupy their potential binding sites in the gut including collagen and fibronectine (Neeser et al. 2000; Lorca et al. 2002). Lactobacillus rhamnosus probiotic strains are of the most thoroughly characterized probiotic bacteria, and therefore LAB represents the most suitable group of bacteria for microbial interference treatment in preventing infectious diseases (Bengmark 1998). The objective of the present work was to study adhesive properties of some potential probiotic LAB strains, among the more representative chicken intestinal isolates (Bouzaine et al. 2003) using the in vitro Basement Membrane Matrigel (BMM) model and to investigate the capacities to adhere and colonize the gastrointestinal tract (GIT) of chicken for a selected strain, L. rhamnosus TB1.

M A T E R I A LS A N D M E T H O D S Bacteria used and molecular identification The strains (five Lactobacillus ssp., two Weissella ssp. and three Enterococcus ssp.) tested in this study represent some of the more representative strains retrieved in a study of gastrointestinal flora in broiler chickens in Tunisia (Bouzaine et al. 2003). Bacillus subtilis 168 and Escherichia coli EPEC (O111) were used respectively as negative and positive controls strains for BMM attachment. Strains were identified previously by biochemical methods. To confirm their identification, a partial 16S rDNA sequence (800 bp) was determined. Preparation of genomic DNA from bacteria, PCR method and the DNA sequences of the PCR products were described by Hyronimus et al. (1998). The 16S rRNA gene fragments were amplified by PCR using the universal primers 20F (5¢-AGAGTTT GATCATGGCTCAG-3¢) and 1500R (5¢-GGTTACC TTGTTACGACTT-3¢) that amplify the maximum number of nucleotides in 16S rDNA from the bacterial taxa. Databases (GenBank) were scanned for sequences similar to the 16S rDNA sequence obtained.

Agar diffusion method for the determination of antibiotic susceptibility patterns Ten antibiotics were tested: penicillin G (10 mg), ampicillin (10 mg), cefaloxin (30 mg), tetracycline (30 mg), chloramphenicol (30 mg), erythromycin (15 mg), spiramycin (10 mg), gentamicin (15 mg), nalidixic acid (30 mg), and rifampicin (30 mg) (disks; Sanofi Diagnostics Pasteur, Marnes la Coquette, France). Instructions from the ‘‘Comite´ Français de l’Antibiogramme’’ related to streptococci were followed (Socie´te´ Française de Microbiologie, Comite´ de l’Antibiogramme 1998). MRS cultures were suspended at approx. 108 CFU ml)1 (McFarland standard 0Æ5). These suspensions were diluted 1 : 100 and 200 ll seeded on Mueller–Hinton agar plates containing 5% sheep blood (bioMe´rieux, Marcy l’Etoile, France) using a swab. Plates were then air dried for 15 min. Disks impregnated with antibiotics were positioned on the plates. After 36 h of incubation at 37C, inhibition zones were recorded. In vitro adherence assays The in vitro adherence of different strains was examined as described by Kukkonen et al. (1993) using BMM (from mouse sarcoma, Becton Dickinson, Le Pont de Claix, France). This preparation contains type IV collagen and laminin as major components followed by heparan sulfate proteoglycans, entactin and nidogen (Horie et al. 2002). The BMM preparation (1/20 dilution) was coated on Lab-Tek Chamber SlidesTM (Nalge Nunc International, Naperville, IL, USA). The slides were then quenched for 2 h at room temperature with 2% BSA-PBS and used in adhesion assays. LAB strains were grown overnight in MRS broth (Difco) and B. subtilis and E. coli strains in Mueller–Hinton broth (Difco) at 37C. Bacteria from early stationary growth phase were collected by centrifugation and washed twice in sterile PBS then stored in the slides at a concentration of 5 · 107 ml)1 and incubated at room temperature for 2 h. Following incubation and subsequent washing of the slides, the adherent bacteria were stained with Gram’s crystal violet solution. Randomly chosen fields were photographed using a light microscopy (Olympus, Rungis, France), the number of bacteria in 12 fields was counted and the results expressed as mean ± SD. Every strain was tested in three different experiments. In vivo adherence assays Culture and labelling of bacteria with carboxyfluorescein diacetate succinimidyl ester (cFDA-SE). Lactobacillus rhamnosus TB1 cells were grown in MRS broth to the early stationary phase (16 h) and then harvested by centrifugation at 9300 g during 5 min, washed twice in PBS (pH

ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 40, 391–396, doi:10.1111/j.1472-765X.2005.01684.x

ADHERENCE AND COLONIZATION OF L. RHAMNOSUS TB1

7Æ5) and resuspended in PBS adjusted to give a cell concentration 1010 CFU ml)1. Lactobacillus rhamnosus TB1 was stained by using a VybrantTM cFDA-SE Tracer Kit (Molecular Probes, Eugene, OR, USA) as described previously by Fuller et al. (2000) and following the manufacturer’s recommended protocols. cFDA-SE is colourless and nonfluorescent until its acetate group is cleaved by intracellular esterase to yield highly fluorescent, amine-reactive carboxyfluorescein succinimidyl ester. The SE group reacts with intracellular amines, forming fluorescent conjugates that are well retained. About 1010 CFU ml)1 of L. rhamnosus TB1 were labelled with 50 lmol l)1 of cFDA-SE at 37C for 2 h. Labelled cells were harvested by centrifugation, washed twice in PBS then resuspended in 200 ml of MRS broth and cultivated at 37C for one generation (c. 2 h). Growth was monitored by measuring OD560 and fluorescent intensities by direct observation using epifluorescence microscopy. The staining efficiency was calculated as follows: % efficiency ¼ (no. of cells stained with cFDASE)/(no. of total cells) · 100. The viable biomass obtained was analysed by plating appropriate sample dilutions onto MRS agar (48 h at 37C). After production, stained cells were harvested by centrifugation resuspended in PBS, protected from light and stored at 4C until their oral inoculation in broiler chickens. Animals. Two broiler chickens 3 weeks old obtained from the Zootechnology Department of Gembloux’s Agronomy Faculty were placed in an experimental cage. Chickens were fasted 12 h before bacteria treatment and cFDA-SE stained L. rhamnosus (109 cells per chicken) were orally inoculated. Following treatment, poultry were supplied with balanced food and water under 12 h light-dark cycles during all experimental assay. Sample preparation from chicken intestinal tract After injection of Zoletil (anaesthetic product; Virbac, Carros, France), the abdominal cavity was opened and various portions of 1 cm of different digestive tract sections (jejunum, ileum, caecum and rectum) were taken. Any visible residual food particles or faecal material were removed. A solution of Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, the Netherlands) was immediately added to each tissue. Samples were submerged in a batch of methanol and dry-ice to allow freezing of Tissue-Tek and then stored at )80C until used for histological preparation. Every sample was scraped in five histological layers measuring from 8 to 10 lm using a micro-cryostat (Slee Benchtop Cryostat, M.T.C., Zaventem, Belgium). Coating slides were covered with aluminium foil and stored at 4C until microscopic observations. To quantify the amount of adherent bacteria, 10 randomly selected microscopic fields were observed from each tissue piece. The area of one

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microscopic field was 3Æ8 · 10)2 mm2. The number of L. rhamnosus-stained bacteria observed was usually between 0 and 22 bacteria by field, considering the addition of bacteria observed in each of the five layers in that each tissue portion was scraped. As a negative control, a similar preparation was performed using a gastrointestinal tissue from chicken that do not received stained bacteria.

RESULTS Identification of LAB strains Bacteria used in this study were identified on the basis of phenotypic and physiological characteristics represented in Table 1 (Bouzaine et al. 2003). Their identification was confirmed by partial 16S rDNA sequencing (800 bp). The 16S rDNA nucleotide identity observed was between 99Æ8 and 99Æ9% with the corresponding species, which were Lactobacillus rhamnosus (TB1), Weissella kimchi (WKT1, WKT2), Enterococcus faecium (EUMT8, EUMT21), Ent. faecalis (EIST1) L. johnsonii (LJT1, LJT2), L. reuteri (LRT1), and L. salivarius (LST1). Antibiotic susceptibility patterns The strain of L. rhamnosus TB1 was sensitive to the majority of the antibiotics tested, including penicillin G, ampicillin, cefaloxin, chloramphenicol, erythromycin, gentamicin, and rifampicin. However, it was resistant to tetracycline, spiramycin, and nalidixic acid. In vitro adherence assay In BMM assay, for the 10 LAB tested two were strongly adherent to Matrigel and the rest showed a weak or nonadherence. Lactobacillus rhamnosus TB1 adhere strongly (c. 2 · 103 cells attached per microscopic field) compared with the positive control, an E. coli EPEC strain. In addition, L. reuteri LRT1 also showed a capacity to adhere but five times less than L. rhamnosus TB1. Lactobacillus salivarius LST1 adhered weakly. The other strains tested were nonadherent. The results are shown in Fig. 1. For W. kimchi (WKT1, WKT2) and Ent. faecium (EUMT8, EUMT21), both strains tested gave similar results (Fig. 1). In vivo cFDA-SE-stained bacteria observations The cFDA-SE-stained bacteria were observed by epifluorescence (green stain). The staining efficiency obtained was up to 90%. In order to quantify adherence of L. rhamnosus TB1 to GIT, the coating slides of the different digestive tract sections were observed by epifluorescence microscopy. The results are expressed as the number of bacteria observed



Table 1 Phenotypic characteristics of strains isolated from gastrointestinal tract of poultry

Characteristics

Number of adhered cells per field

Gas from glucose Growth at 45C Growth at 15C Growth at 4% NaCl Growth at 6Æ5% NaCl Growth at 8% NaCl Ammonia from arginine Fermentation of Arabinose Cellobiose Esculin Fructose Glucose Lactose Mannose Melibiose Raffinose Rhamnose Ribose Salicin Trehalose Xylose

L. reuteri LRT1

L. johnsonii LJT2

W. kimchi WKT1

L. salivarius LST1

L. rhamnosus TB1

Ent. faecalis EIST1

Ent. faecium EUM8

+ ) ) + + + +

) ) ) + + + )

+ ) ) + ) ) )

) ) ) + + + )

) ) ) + + + )

) + ) + + + +

) + ) + + + +

+ ) ) ) + + ) + + ) + ) ) +

) + + + + + + ) + ) ) ) + )

+ + + + + ) + ) ) ) + ) ) +

) ) + + + + ) ) + ) ) + + )

) + + + + + + ) ) + + + + )

) + + + + + + + + ) + + + )

) + + ) + + + + + ) + + + )

Table 2 Adhesion of Lactobacillus rhamnosus TB1 to the chicken gastrointestinal tract sections

3500 3000

No. of bacteria observed per microscopic field (3Æ8 · 10)2 mm2)*

2500 2000

Intestinal sections

3h

Jejunum Ileum Caecum Rectum

10 11 1 17

24 h

1500 1000 500

BS 16 8 EC 01 11

Strains

TB 1

LJ T2 LR T1 LS T1

W KT 1 EU M T8 EI ST 1

0

Fig. 1 Adhesion of LAB isolated from gastrointestinal tract of poultry to basement membrane Matrigel. Means values and SD of bacterial numbers in 12 random microscopic fields observed by oil immersion (·1000). Strains – WKT1: Weissella kimchi WKT1; EUMT8: Enterococcus faecium EUMT8; EIST1: Ent. faecalis EIST1; LJT2: Lactobacillus johnsonii LJT2; LRT1: L. reuteri LRT1; LST1: L. salivarius LST1; TB1: L. rhamnosus TB1; BS168: Bacillus subtilis 168; EC O111: Escherichia coli O111

by microscopic field (3Æ8 · 10)2 mm2). In contrast, no stained bacteria or green fluorescence were detected in tissues from the control group.

± ± ± ±

4 2 3 4

10 10 1 14

± ± ± ±

3 5 4 3

*Addition of bacterial number observed in the five scraped layers corresponding to the same tissue portion.

Three hours after the inoculation the most important number of cFDA-SE-labelled bacteria was localized in rectum, 17 ± 4 bacteria per tissue surface of 3Æ8 · 10)2 mm2. Moreover an important number was also observed in the jejunum and ileum, 10 ± 4 and 11 ± 2 bacteria respectively, per tissue surface of 3Æ8 · 10)2 mm2 (Table 2). No significant decrease in the number of adherent L. rhamnosus TB1 was observed 24 h after bacterial inoculation (Table 2). Furthermore, staining bacteria are specific to certain intestinal zones, in fact, the caecal villi contain the most of the bacteria attached in caecum.


ADHERENCE AND COLONIZATION OF L. RHAMNOSUS TB1

DISCUSSION Adhesion to the intestinal mucosa could confer a competitive advantage, important for bacterial maintenance in the GIT and it is generally accepted that adhesion properties contributes to the efficacy of probiotic strains (Bernet et al. 1994; Hudault et al. 1997; Servin and Coconnier 2003). Therefore, we analysed the adherent capacity of some LAB strains that were previously selected as a potential strains for probiotic design based on their antimicrobial activity and resistance to the bile and gastric acidity (Bouzaine et al. 2004). In the present study we have shown that some lactobacillus strains isolated from the chicken GIT can adhere to the Matrigel. Among the LAB tested, L. rhamnosus TB1 was the strain that presented the most adherent capacity in vitro. In order to confirm the adherence properties of L. rhamnosus TB1 in GIT of broilers, an in vivo adherence assay was undertaken using cFDA-SE bacterial staining method. This method was previously proposed to be used for the microbial ecology research. Moreover, Fuller et al. (2000) reported that cFDA-SE was able to label cells without compromising cell viability or altering cell adhesion characteristics. Recently, Lee et al. (2004) realized a study to investigate the colonization potential of L. casei Shirota in mouse intestine using the cFDA-SE staining. Lactobacillus rhamnosus TB1 showed a good ability to adhere to intestinal epithelial cells of chicken with a good affinity to rectal tissues, ileum and jejunum. According to Fuller et al. (2000) during the growth of stained bacteria, the number of cells would be expected to increase, while the number of detectable CFDA-staining cells would be expected to decrease due to dilution of the intracellular CFDA concentration after each cell division. Therefore, to avoid the fluorescent marker dilution, chicken tissues were analysed only 3 and 24 h after the bacterial inoculation. Jin et al. (1996) reported the ability of some Lactobacillus isolates to adhere strongly to the chicken ileal epithelial cells. Bacterial attachment to the epithelial wall of the ileum probably controls the intestinal flora of this region, and it may be one of the defence mechanism against the colonization of the small intestine by other undesirable bacteria from the caecum (Sarra et al. 1992). Further studies on the capacity of lactobacilli to colonize the chicken crop using in vivo experiments are needed to investigate probiotic administration as a biological alternative for the use of antibiotics as growth promoters for poultry and other food animals. In fact, information about the mechanism involved in the adherence of lactobacilli to the GIT may eventually help producers of dietary suppliants to make more rational decisions on the lactobacilli strain selection during design of the commercial products. Several studies demonstrated that competition between pathogenic and ‘beneficial’ bacteria to

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the site of adhesion may play an important role in the host protection against infection. However, recent studies suggested that probiotic adhesion was not necessary in order for these bacteria to exert their effect in the GIT. This was suggested to be caused by the specificity of attachment site and the removal of mucus (Bezkorovainy 2001). Moreover, Hautefort et al. (2000) also reported that adhesion was not sufficient to ensure the persistence of L. fermentum strains in the digestive tract of mice. As adhesion may lead for a prolonged residence of probiotic bacteria in the intestinal tract, such a property was also considered as a potential virulence factor involved in the translocation of pathogens across the mucosal surface (Finlay and Falkow 1997). Lactic acid bacteria display various surface determinants that are involved in their interaction with intestinal epithelial cells. Servin and Coconnier (2003) recently showed that the LAB adhesion process includes passive forces, electrostatic interaction, hydrophobic, steric forces. It has been reported that lipoteichoic acids are one of the factors responsible for adhesion of L. johnsonii LaI (Granato et al. 1999). In addition, adhesion-promoting proteins, which are present on the cell surface of L. fermentum and L. brevis has been isolated and characterized (Jakava-Viljanen et al. 2002; Lorca et al. 2002; Rojas et al. 2002). The adhesion process may also involve interaction of bacterial compounds such as lectins with the surface of the intestinal mucosa (Adlerberth et al. 1996; Neeser et al. 2000). Lactobacillus rhamnosus TB1 is able to agglutinate yeast cells in a mannose-sensitive manner (data not shown). In consequence it is possible that this strain posses adhesins specific for mannose receptors (Adlerberth et al. 1996). Antibiotic susceptibility patterns vary greatly between different species of lactobacilli. Moreover, strains with atypical resistance pattern, which include resistance to some clinically important antibiotics have been detected among lactobacilli (Charteris et al. 1998). The last finding suggests that antibiotic resistance pattern should be tested for each particular probiotic strain. Our strain, sensitive to most of the antibiotics, is tetracycline resistant. Future experiments should investigate whether this resistance is carried by a resistance plasmid and can be transmitted to other bacteria. In conclusion, the present study demonstrates that adherence to BMM is an easy method for screening LAB in order to assess in vitro adhesion properties of the probiotic bacteria. Moreover, the L. rhamnosus TB1 strain was not excluded and was able to attach in vivo to epithelial tissue of the different sections of GIT (jejunum, ileum and rectum) after its inoculation in vivo. Previous studies also showed that this strain possessed some antimicrobial activity and was resistant to bile and gastric acid treatments (Bouzaine et al. 2004). Based on our study, we concluded that L. rhamnosus TB1 strain may be considered as a potential



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