Sarmiento-Rubiano et al. BMC Genomics 2010, 11:504 http://www.biomedcentral.com/1471-2164/11/504 immunogenicity of an oral human rotavirus vaccine in animals [6]. L. acidophilus strain Bar13 interfered with the adhesion of enteropathogenic bacteria to enterocytes and inhibited IL-8 production by HT-29 cells, suggest- ing a potential to protect intestinal cells from acute inflammatory response [7]. L. johnsonii strain La1 (NCC533) has been shown to suppress gene expression of proinflammatory cytokines, supposedly involved in atopic dermatitis [8,9] and acts as an immunomodulator [10-12]. The genome of L. johnsonii strain NCC533 has been sequenced [13] and has enabled a number of stu- dies aimed to determine the genes which are expressed in vivo and those whose expression is specifically neces- sary for long persistence in the gut [14,15]. The bacterial species concept has been widely dis- cussed [16,17], but no consensus has been reached to establish a regularly acceptable definition. In the case of prokaryotic pathogens, species are traditionally identified on the basis of the disease they caused, regardless of genetic considerations. However, for the vast majority of bacteria, phenotypic characteristics are generally not as precise as identification based on genotypic methods. The observation that there are a fraction of genes found in bacterial genomes shared by all members of a species and a fraction present only in a subset of the population gave rise to the core genomic hypothesis [18]. It postu- lates that there is a core of genes responsible for keep- ing a species identity and auxiliary genes responsible for gene transfer and adaptation of strains to the environ- ment. The incorporation of molecular data permitted to state that a bacterial species is “a category that circum- scribes a (preferably) genomically coherent group of individual isolates/strains sharing a high degree of simi- larity in (many) independent features, comparatively tested under highly standardised conditions” [19]. Essen- tially, a species would be a group of strains with certain level of phenotypic consistency and showing DNA-DNA re-association rates greater than 70% [19]. However, this variability cut-off method is not always appropriate due to the fact that different clearly recognized species have a large range of genetic variation. Since DNA sequen- cing is nowadays an accessible technique to most laboratories, comparative sequence analysis of the 16S rRNA gene has been extensively used for species identi- fication in novel isolates [20]. The ad hoc committee for the re-evaluation of the species definition in bacteriology considered sequencing of housekeeping genes, DNA profiling and DNA arrays as very valuable methods for species delineation and phylogenetic positioning [19]. The L. acidophilus group constitutes a paradigm of a compact cluster showing closely related species that offers special difficulties for the correct taxonomic allo- cation of new strains. Several techniques have been applied to identify members of the L. acidophilus group, Page 2 of 16 such as 23S-rRNA-targeted oligonucleotide probe hybri- dization [21], random amplification of polymorphic DNA [22,23], sequence alignment of the V1 region of the 16S rRNA encoding gene [24] and automated ribo- typing [25]. Recently, more complex genomic techniques based on multilocus sequence analysis of five house- keeping genes and comparative genome hybridization (CGH) using microarrays have been successfully applied to the L. acidophilus complex [26]. They showed that L. johnsonii, L. gasseri and L. acidophilus constitute three clearly independent and consistent genomic groups, although L. gasseri was more closely related to L. johnso- nii than to L. acidophilus. The bacterial genomic varia- bility between strains belonging to the same species or between closely related species comes from mobile DNA elements and from variable regions. The latest may be required for environmental adaptation and could be acquired by lateral gene transfer. The L. john- sonii NCC533 chromosome harbors three regions that contain two prophages, Lj928 and Lj965, and a 6-kb potentially autonomous unit [27,28]. These genetic ele- ments represent more than half of the identified strain- specific DNA and have been extensively studied [26-28]. L. johnsonii NCC533 also carries four further integrases and several IS elements flanking or disrupting diversity regions [26-28]. A previous work studying the influence of sorbitol intake on the population of rat intestinal lactobacilli showed the presence of five species: L. johnsonii, Lacto- bacillus intestinalis, Lactobacillus murinus, Lactobacillus reuteri and Lactobacillus sp. BL263 (previously named as AD102) [29]. Sorbitol ingestion resulted in an incre- ment of L. reuteri and Lactobacillus sp. BL263 cell num- bers [29]. Although strain Lactobacillus sp. BL263 could be assigned to the L. acidophilus group, its species allo- cation was still uncertain. In the present study, standard taxonomic methods and microarray-based CGH analysis have been implemented for the identification and char- acterization of Lactobacillus sp. BL263 and four addi- tional rat intestinal isolates (BL301, BL302, BL303 and BL304) displaying identical PCR-DGGE pattern. Methods Bacterial strains, culture conditions, DNA extraction The Lactobacillus strains used in this work are listed in Table 1 and they were grown in MRS medium (Difco) at 37°C during 48 h under static conditions. Chromoso- mal DNA was isolated as described before [30]. 16S rRNA encoding gene and multilocus sequence analyses The housekeeping genes selected for multilocus sequence analysis based on the results of previous stu- dies on Lactobacillus species [26,31] were recombinase