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Applied and Environmental Microbiology, July 2007, p. 4385-4388, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.02470-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Screening of Exopolysaccharide-Producing Lactobacillus and Bifidobacterium Strains Isolated from the Human Intestinal Microbiota{triangledown}

Patricia Ruas-Madiedo,* José Antonio Moreno,{dagger}, Nuria Salazar, Susana Delgado, Baltasar Mayo, Abelardo Margolles, and Clara G. de los Reyes-Gavilán

Departamento de Microbiologia Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias, CSIC, Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain

Received 23 October 2006/ Accepted 26 April 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 REFERENCES
 
Using phenotypic approaches, we have detected that 17% of human intestinal Lactobacillus and Bifidobacterium strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
REFERENCES
 
The human intestinal microbiota has around 10 times more cells than the human body (2), and its genome ("microbiome") harbors at least 100 times more genes than our own genome (5). However, a significant fraction of intestinal bacteria has not been described yet, making it difficult to understand the mechanisms of communication among the microbiota, host cells, and intestinal environment (21). Bifidobacterium and Lactobacillus species are common inhabitants of the gastrointestinal tract, and they have received special attention because of their long history of safe use in foods and probiotic effect (11). Some probiotic strains are able to adhere to intestinal mucus, and we have postulated that exocellular polysaccharides isolated from lactic acid bacteria (LAB) and bifidobacteria interfere with the adhesion of probiotics and pathogens to human intestinal mucus (9, 10). Therefore, the production of exopolysaccharides (EPS) could also be an interesting property to consider for the selection of putative probiotic strains (17). Currently, the most suitable approach to the search for novel EPS-producing (EPS+) strains is the exploration of wild bacteria (6). The aim of this study was to investigate the EPS+ capabilities among Lactobacillus and Bifidobacterium strains isolated from the human intestinal ecosystem as the initial step for further investigation of the roles of EPS in bacterium-host interactions and in human health.

We have employed 362 Lactobacillus and Bifidobacterium strains, previously isolated from fecal and mucosal samples of healthy adult volunteers (3, 4), and 4 reference EPS+ bifidobacteria (Bifidobacterium animalis subsp. lactis IPLA-R1, Bifidobacterium longum NB667 [10], B. longum BL1, and B. longum 667Co) for screening of EPS production in solid media. Strains were grown at 37°C for 72 h under anaerobic conditions on the surface of MRS agar containing 0.25% L-cysteine (MRSC agar) and supplemented with 2% glucose, fructose, lactose, or sucrose added separately. Sixty putative EPS+ strains were detected, most of them being mucoid (92%) and only 5 (8%) having a "ropy" character. The percentage of putative EPS+ strains (17%) was similar to that reported in the literature for EPS+ LAB isolated from food environments (1, 6, 12) and from animal origins (14). Amplification, sequencing, and comparison with database sequences for the V1-V2 variable region of the 16S rRNA gene (7, 19) identified 35 out of our 60 putative EPS+ strains as belonging to the genus Bifidobacterium and 25 as belonging to Lactobacillus (with nucleotide identity at the species level higher than 98%). The EPS+ bifidobacteria were Bifidobacterium pseudocatenulatum (51%), B. longum (40%), B. animalis (6%), and Bifidobacterium adolescentis (3%). These results were in general coincident with those for the most abundant species found in the fecal samples of the donors (4). The EPS+ lactobacilli were Lactobacillus casei (48%), Lactobacillus rhamnosus (24%), Lactobacillus plantarum (16%), Lactobacillus gasseri (4%), Lactobacillus acidipiscis (4%), and Lactobacillus vaginalis (4%). Contrary to what was found for bifidobacteria, L. gasseri was the most abundant lactobacilli in the intestinal population of donors but one of the lowest EPS producer species. Remarkably, all L. plantarum human isolates were putative EPS+ strains.

A screening for glycansucrase and glycosyltransferase genes related to the synthesis of homo- and heteropolysaccharides, respectively, was performed on the 60 putative EPS+ strains of human origin and on the 4 reference bifidobacteria. PCR techniques were employed to detect fragments of genes coding for glucansucrases (14) and fructansucrases (13) as well as for the glycosyltransferase involved in the synthesis of ß-D-glucan (20), but the results obtained were not positive. The pGT primers were employed to amplify the genes coding for the "priming glycosyltransferase," which catalyzes the transfer of a sugar-1-phosphate to a lipophilic carrier molecule anchored in the cellular membrane, this being the first step for the assembly of the repeating unit that builds many heteropolysaccharides (8). Amplifications were obtained for 25 human isolates (11 bifidobacteria and 14 lactobacilli) and the 3 reference strains of B. longum. The predicted amino acid sequences of the PCR products presented high identity with the partial C-terminal regions of the glycosyltransferases (Fig. 1). Those of the bifidobacteria showed identity (83% to 95%) with two sequences from B. longum NCC2705, those of the undecaprenyl-phosphate sugar phosphotransferase RfbP and the galactosyltransferase CpsD. Most sequences from the Lactobacillus strains showed identity (93% to 100%) with that of the putative undecaprenyl-phosphate glycosyl-1-phosphate transferase from L. rhamnosus or with that of the priming glycosyltransferase of Lactobacillus paracasei. Within all these sequences, parts of blocks B and C involved in the interaction with the lipid carrier and in the recognition of the sugar specificity, respectively, were found (8, 18). A glutamate residue present in block C (8) has been proposed as one of the candidates for the catalytic residues in the priming glycosyltransferase of Lactococcus lactis (16). Finally, the 28 sequences of our EPS+ strains showed a tyrosine in block C which is conserved in the sequences of priming galactosyltransferases but not in those of glucosyltransferases (16). All these data suggest that our EPS+ strains can carry genes involved in the synthesis of heteropolysaccharides. A phylogenetic tree was constructed with these amino acid sequences (Fig. 2), including some sequences from LAB and bifidobacteria reported in databases. The glycosyltransferase sequences of lactobacilli and bifidobacteria clustered separately. All strains of B. pseudocatenulatum and the strain B. longum L56 were closely related to the RfbP sequence of B. longum NCC2705. The remaining B. longum strains, including the reference strains, were more related to the CpsD sequence from B. longum NCC2705. The sequences of our L. casei and L. rhamnosus strains were grouped in several closely related clusters and appeared more differentiated from other species of this genus. L. rhamnosus strains E41, E42, and E43R isolated from the same individual were similar to group 2 of the classification by the sequence homology of Provencher and coworkers (8), but none of our isolates fit in their group 1 category. The L. rhamnosus strains G93 and G94 were closely related to the sequence of L. paracasei type V and also with some intestinal strains of L. casei. However, L. rhamnosus G92, isolated from the same individual as G93 and G94, appeared clearly differentiated from the other intestinal L. rhamnosus and L. casei strains. Our results showed variability in the sequences of glycosyltransferases among strains isolated from different individuals and differences among strains from the same individual. Divergences of priming glycosyltransferase sequences among closely related microorganisms have been previously reported by other authors, and more than one potential priming glycosyltransferase can be present in a unique strain (8).


Figure 1
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  		FIG. 1. Alignment of an internal fragment close to the C-terminal amino acid sequences (44 amino acids each) of priming glycosyltransferases deduced from PCR amplifications with pGT primers for 28 strains employed in this study: 25 of human origin (marked with one asterisk) and 3 from the reference strains Bifidobacterium longum NB667 (NIZO food research culture collection), B. longum 667Co, and B. longum BL1 (IPLA culture collection) (marked with two asterisks). The internal fragments close to the C-terminal amino acid sequences for some LAB and bifidobacterial strains held in databases are also included in the alignment. The GenBank accession numbers of all strains are shown in parentheses. The shading identifies amino acids that are identical (dark gray) or conserved (light gray). The letter X indicates an undetermined amino acid. Arrows show the amino acid residues glutamate (E) and tyrosine (Y).

 

Figure 2
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  		FIG. 2. Phylogenetic tree of an internal fragment close to the C-terminal amino acid sequences (44 amino acids each) of priming glycosyltransferases obtained by PCR amplifications with pGT primers for 28 strains employed in this study: 25 from human origins (marked with one asterisk) and 3 from the reference strains Bifidobacterium longum NB667 (NIZO food research culture collection), B. longum 667Co, and B. longum BL1 (IPLA culture collection) (marked with two asterisks). The GenBank accession numbers of the strains from this study are included in Fig. 1. Sequences of genes from 28 LAB and bifidobacteria held in databases were also included in the phylogenetic analysis, and their GenBank accession numbers are shown in parentheses. The numbers associated with the branches indicate the bootstrap values (confidence limits) resulting from 500 replicate resamplings. The bar scale refers to the number of amino acid substitutions per site.

 
Finally, 2 reference strains and 21 putative EPS+ isolates,originating from different individuals and selected on the basis of their differential carbohydrate fermentation patterns (data not shown), were employed in order to demonstrate their abilities to synthesize EPS (Table 1). EPS fractions were isolated from the surfaces of cultures on MRSC agar (10) to avoid the coisolation of glucomannans present in the medium (15). No correlation between the production of EPS and amplification of the pGT primers was obtained for any L. plantarum or B. animalis strain and for half of the B. longum strains. However, there was a positive correlation between both parameters for the L. rhamnosus and L. casei groups. Since the pGT primers were designed based on the codon usage of L. rhamnosus (8), this result was expected.


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  		TABLE 1. Amplification of priming glycosyltransferase genes by PCR and isolation of the EPS fractions produced by selected bifidobacteria and lactobacilli

 
In short, the gastrointestinal tract can be a good environment for the isolation of novel heteropolysaccharide-producing Lactobacillus and Bifidobacterium strains. The degenerated pGT primers (8) could be used to detect some of these strains. However, not all intestinal EPS+ strains can be evidenced with these primers, probably due to sequence heterogeneity of priming glycosyltransferases from different microorganisms.


   ACKNOWLEDGMENTS
 
This work was financed by FEDER funds (European Union) and the Spanish Plan Nacional de I+D+I through project AGL 2004-06088-CO2-01/ALI and by a grant from the Fundación Príncipe de Asturias (Beca Grande Covián 2003). P. Ruas-Madiedo and J. A. Moreno were the recipients of an I3P postdoctoral research contract granted by CSIC. S. Delgado and N. Salazar acknowledge the Spanish Ministry of Education and Science for their respective fellowships (Programa FPI).

Our thanks to P. López and M. L. Werning (CIB-CSIC) for sharing the primers designed from the ß-glucan glycosyltransferase nucleotide sequence before publication.


   FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Productos Lácteos de Asturias, CSIC, Ctra. Infiesto s/n (P.O. 85), 33300 Villaviciosa (Asturias), Spain. Phone: 34-985892131. Fax: 34-985892233. E-mail: ruas-madiedo{at}ipla.csic.es Back

{triangledown} Published ahead of print on 4 May 2007. Back

{dagger} Present address: Laboratorios Ordesa, Carretera del Prat 9-11, 08830 Sant Boi Llobregat, Spain. Back


   REFERENCES
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Applied and Environmental Microbiology, July 2007, p. 4385-4388, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.02470-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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