CHARRIER ET AL. Biol Res 39, 2006, 669-681 669 Biol Res 39: 669-681, 2006 BR Isolation and characterization of cultivable fermentative bacteria from the intestine of two edible snails, Helix pomatia and Cornu aspersum (Gastropoda: Pulmonata) MARYVONNE CHARRIER*, 1, GÉRARD FONTY2, 3, BRIGITTE GAILLARD-MARTINIE2, KADER AINOUCHE1 and GÉRARD ANDANT2 1 Université de Rennes I, UMR CNRS 6553 EcoBio, 263, Avenue du Général Leclerc, CS 74205, F-35042 Rennes Cedex, France. 2 Unité de Microbiologie, CR INRA de Clermont-Ferrand - Theix, F-63122 Saint Genès-Champanelle, France. 3 Laboratoire de Biologie des Protistes, UMR CNRS 6023, Université Clermont II, 63177 Aubière, France. ABSTRACT The intestinal microbiota of the edible snails Cornu aspersum (Syn: H. aspersa), and Helix pomatia were investigated by culture-based methods, 16S rRNA sequence analyses and phenotypic characterisations. The study was carried out on aestivating snails and two populations of H. pomatia were considered. The cultivable bacteria dominated in the distal part of the intestine, with up to 5.109 CFU g -1, but the Swedish H. pomatia appeared significantly less colonised, suggesting a higher sensitivity of its microbiota to climatic change. All the strains, but one, shared ≥ 97% sequence identity with reference strains. They were arranged into two taxa: the Gamma Proteobacteria with Buttiauxella, Citrobacter, Enterobacter, Kluyvera, Obesumbacterium, Raoultella and the Firmicutes with Enterococcus, Lactococcus, and Clostridium. According to the literature, these genera are mostly assigned to enteric environments or to phyllosphere, data in favour of culturing snails in contact with soil and plants. None of the strains were able to digest filter paper, Avicel cellulose or carboxymethyl cellulose (CMC). Acetogens and methanogenic archaea were not cultivated, so the fate of hydrogen remains questionable. This microbiota could play important roles in the digestive process (fermentation) and the energy supply of the snail (L-lactate, acetate). The choice of cereals and plants by snail farmers should take into account the fermentative abilities of the intestinal microbiota. Key terms: Clostridium, edible snails, Enterobacteriaceae, Enterococci, gut microbiota, Lactococci INTRODUCTION cellulose, laminaran and mannan are highly degraded by Helicid snails while starch, Terrestrial gastropods feed upon fresh pullulan, α-glucosides, xylan and β- plants with high protein and calcium xylosides are weakly hydrolysed. contents (Iglesias and Castillejo, 1999; The dependence of pulmonates on Chevalier et al.,2003) and may also microbial activity within their gut would participate, with other soil invertebrates, in explain their extraordinary efficiency in the decomposition of leaf litter plant fibre digestion (60-80%) (Davidson, (Hatziioannou et al., 1994). Consequently, 1976; Charrier and Daguzan, 1980). A they need a large set of polysaccharide survey of the dominant cultivable bacteria in depolymerases and glycoside hydrolases for the digestive tract of snails revealed mainly the digestion of plant materials. Previous Enterobacteria (Lesel et al., 1990; Watkins works (Myers and Northcote, 1958; and Simkiss, 1990) and Enterococci Charrier and Rouland, 1992; Flari and (Charrier et al., 1998). Some authors Charrier, 1992) demonstrated that native suggested the role of bacteria in fermentative Corresponding author: Tel: +(33)2.23.23.50.45; Fax: +(33)2.23.23.50.26; E-mail: [email protected] Received: March 29, 2006. In revised form: July 21, 2006. Accepted: July 25, 2006 670 CHARRIER ET AL. Biol Res 39, 2006, 669-681 processes of the hosts (Charrier et al., 1998) originated from Luberon (southern France) in particular the breakdown of chitin and was collected in a garden covered with soluble cellulose (Lesel et al., 1990; Poaceae, Trifolium repens and Taraxacum Brendelberger, 1997). Others argued that sp. Helix pomatia was taken from two bacteria are transient populations able to distinct populations, one living in Götaland interact with snail intake of soil pollutants (southern Sweden) and the other in (Simkiss, 1985; Simkiss and Watkins, 1990). Auvergne (central France). They lived on Only recently were described the mixtures of meadows and woods that environmental characteristics (pH, O2) in consist of deciduous trees (oaks, beechs) Helicid guts (Charrier and Brune, 2003). The and herbaceous plants (Poaceae, Urtica authors revealed a pH gradient along the gut, dioïca, Taraxacum sp., Ranunculus sp. in from acidic in the crop to neutral or alkaline Auvergne, Aegopodium podagraria, in the intestine. The anoxic status of the Mercuralis perennis and Poa nemoralis in whole gut was also demonstrated. Götaland). The snails were collected in Unfortunately, most of the microbiological aestivation during summer. data concerned aerobically cultivable bacteria and focused on crop fluids and Preparation of the intestinal extracts digestive gland-intestine mixed extracts. Moreover, the breakdown of polysaccharides The snails were anaesthetised with 0.1 mM and oligosaccharides predominates in the succinyl chloride in 2% MgCl2 (Chung, anterior tract (oesophagus, crop and 1985). They were dissected in five sets of stomach) where bacteria appeared mainly three (H. pomatia) or six (C. aspersum) aerobic, at the level of 1.3 106 CFU g-1 tissue individuals inside an anaerobic glove box, (Charrier et al., 1998). In contrast, the as previously described (Charrier et al., bacterial densities stay at a high level (2 108 1998). The intestine, embedded with the CFU g-1 tissue) in the intestine of fed and flat lobe of the digestive gland, comprised starved animals. two parts defined on histological features. A better knowledge of the roles of the The proximal intestine (PI) ended along the intestinal microbiota in snail nutrition is kidney base and the distal intestine (DI) needed and data should improve intensive was pressed between the glandular acini rearing of these animals. Consequently, the and emerged by the rectum. They were aims of the present paper were: (i) to isolate illustrated elsewhere (Charrier and Brune, the predominant cultivable bacteria of the 2003). For the cultivation experiments two edible snail species Helix pomatia and (three sets of snails), PI and DI were Cornu aspersum (Syn: Helix aspersa), (ii) separately placed into screw-capped tubes to identify the strains at the species or containing the anaerobic mineral solution of genus level using phylogenetic analysis of Bryant and Burkey (1953). For the study of 16S rRNA sequences, and (iii) to determine plant fermentation (two sets of snails), PI the biochemical profiles of the strains. In and DI were put together into flasks order to assess the influence of the containing the mineral solution with environment on the bacterial densities and sterilized plant fragments. communities, the species Helix pomatia was investigated from two populations of Cultivation experiments different geographical origins. (i) Anaerobic counts. All the cultures were performed in triplicate 500 μl aliquots, MATERIAL AND METHODS according to the anaerobic technique of Bryant (1972). Serial decimal dilutions of Snails the gut extracts were prepared under O2 - free CO2 in the mineral solution. Two species of snails were used: the brown A complete solid medium (Leedle and garden snail Cornu aspersum and the Hespell, 1980) was used at pH 7 to quantify vineyard snail Helix pomatia. C. aspersum, the total viable counts of bacteria as the CHARRIER ET AL. Biol Res 39, 2006, 669-681 671 number of colony forming units (CFU). The to the following criteria: morphology, medium, outgassed with a nitrogen-carbon gram staining, motility, 16S rRNA dioxide mixture (80: 20%) was dispensed sequencing, biochemical and nutritional into 16 x 150 mm Hungate tubes sealed characteristics. with butyl rubber stoppers (Bellco Glass In Vineland NJ). They were incubated for 7 Phylogenetic analysis days at 25°C before enumeration. The values were expressed in log CFU g-1 fresh (i) 16S rRNA sequencing. The purified weight ± SD and compared by Kruskal- strains were sent, in 50% glycerol, to Wallis (KW) and Tukey non parametric Macrogen (Seoul, Korea) for sequencing. tests. The 16S r RNA genes were amplified by Cellulolytic bacteria were counted in the PCR using the bacterial primers 27f (5’ - RGCA medium (Fonty et al., 1989) without AGAGTTTGATCCTGGCTCAG- 3’) agar, in which glucose and cellobiose were complementary to position 8-27 (E. coli replaced by cellulose as a strip of filter paper numbering) and 1492r (5’ - (Whatman N° 1). Methanogenic archaea GGTTACCTTGTTACGACTT- 3’) were counted in Balch tubes in the liquid complementary to position 1510-1492 (E. medium of Balch et al., (1979) under H2 / coli numbering). These primers were CO2 (80: 20, v/v, 202 kPa). The presence of described by Lane (1991). Sequence methanogenic archaea in each tube was assemblies were obtained with the CAP determined by GC analysis of CH4 program (http: //www.infobiogen.fr) and production and the cultures were observed pairwise sequence comparisons to 16S with epifluorescence microscopy. rRNA available in DNA sequence databases Acetogenic bacteria were enumerated on the (Genbank, EMBL, DDBJ and PDB) were AC-11 liquid medium (Breznak and Switzer, performed using BLASTN 2.2.10 program 1986) according to the method described by (http: //www.ncbi.nlm.nih.gov/). The Morvan et al., (1996). The media for sequences were edited and aligned cellulolytic bacteria, methanogens and manually using the Bioedit