Pablo Álvarez-Martín

Diversity and evolution of the microbial populations during manufacture and ripening of Casín, a traditional Spanish, starter-free cheese made from cow's milk

Classical culturing and denaturing gradient gel electrophoresis (DGGE) techniques have been used for studying the microbial diversity and dynamics of the traditional Spanish Casín cheese during manufacturing and ripening. As with other starter-free cheeses made from raw milk, the microbial diversity of Casín was shown to be high by both culturing and DGGE analyses. The culture technique showed that lactic acid bacteria (LAB) species constituted the majority of the microbial populations. Of the 14 bacterial species identified, Lactococcus garvieae was predominant in the three-day-old cheese sample, although it was replaced by Lactococcus lactis subsp. lactis at day 30. As expected, the DGGE profiles obtained were complex, consisting, depending on the sample, in five to ten different amplification bands. Among these, a band corresponding to Streptococcus thermophilus was observed throughout the whole manufacturing process. This species had never been identified from traditional Spanish cheeses previously. Culturing and molecular methods showed high populations of undesirable microorganisms, arguing for a required improvement in the hygiene of Casín manufacture. Random amplification of polymorphic DNA (RAPD) profiling suggested that the L. garvieae and L. lactis populations were composed of one and five strains, respectively. In addition, only a single L. lactis RAPD pattern was stably maintained from day three to day 30, indicating high succession of strains along ripening. After a thoroughly characterisation, strains of the two Lactococcus species could be used in designing specific starter cultures for Casín. Additional species (such as Lactobacillus plantarum and Corynebacterium variabile) might be included as adjunct cultures.

Molecular Analysis of tet(W) Gene-Mediated Tetracycline Resistance in Dominant Intestinal Bifidobacterium Species from Healthy Humans

ABSTRACT tet(W) was found responsible for tetracycline resistance (MICs, 4 to ≥32 μg ml−1) in dominant bifidobacterial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66 proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio fibrisolvens.

Analysis of tetracycline resistance tet(W) genes and their flanking sequences in intestinal Bifidobacterium species

OBJECTIVES The tet(W) gene provides tetracycline resistance to a wide range of anaerobic intestinal and ruminal bacteria, but little is known about the molecular organization of the tet(W) gene. The aim of this study was to gain new insights into the molecular organization of the tet(W) gene in bifidobacteria strains from humans. METHODS A segment of DNA encompassing the whole tet(W) gene and its immediate upstream and downstream sequences was analysed in 10 representative strains of four Bifidobacterium species, of which two have been shown to be tetracycline-susceptible. The non-conserved flanking regions of the tet(W) gene were further analysed in six strains. RESULTS All 10 strains share a core DNA domain of 2154 bp [starting 250 bp upstream of the tet(W) gene start codon and ending 13 bp before the stop codon] with 98% to 100% DNA identity. Except for Bifidobacterium animalis E43, all other strains further share 408 bp upstream and 70 bp downstream of the tet(W) gene. An insertion-like element of 736 bp was found to interrupt the tet(W) coding sequence in Bifidobacterium longum M21, which may be the reason for its tetracycline susceptibility. However, genetic events explaining the susceptible phenotype of B. longum LMG 13197(T) were not observed. CONCLUSIONS The tet(W) genes from all 10 strains shared 98% to 100% DNA and amino acid identity, though large variation was found in their flanking regions.

Improved Cloning Vectors for Bifidobacteria, Based on the Bifidobacterium catenulatum pBC1 Replicon

ABSTRACT This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 × 101 to 1.0 × 105 transformants μg−1 but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZα peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an α-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in α-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.

A single mutation in the gene responsible for the mucoid phenotype of Bifidobacterium animalis subsp. lactis confers surface and functional characteristics

ABSTRACT Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.

Controlled gene expression in bifidobacteria by use of a bile-responsive element

ABSTRACT The promoter activity of the upstream region of the bile-inducible gene betA from Bifidobacterium longum subsp. longum NCC2705 was characterized. DNA fragments were cloned into the reporter vector pMDYAbfB, and the arabinofuranosidase activity was determined under different in vitro conditions. A segment of 469 bp was found to be the smallest operational unit that retains bile inducibility. The reporter activity was strongly affected by the presence of ox gall, cholate, and conjugated cholate, but not by other bile salts and cell-surface-acting compounds. Remarkably, this bile-inducible system was also active in other bifidobacteria containing betA homologs.

Candida cabralensis sp. nov., a yeast species isolated from traditional Spanish blue-veined Cabrales cheese.

Three yeast strains, 1AD8(T), 3AD15 and 3AD23, belonging to a previously unknown yeast species were isolated from two independent batches of the Spanish blue-veined Cabrales cheese, a traditional cheese manufactured without the addition of starter and mould cultures. Physiological characterization revealed that the unknown yeast is not fermentative and does not assimilate lactose; rather it assimilates dl-lactic acid and ethanol, major end products of lactic acid bacteria metabolism in cheese. The novel yeast is anamorphic. Phylogenetic tree reconstruction based on nucleotide sequence comparison of the D1/D2 region of the 26S rRNA gene showed that Pichia terricola and Pichia fermentans are the closest relatives of the unknown species. The name Candida cabralensis sp. nov. is proposed, and the isolate 1AD8(T) (=CECT 13027(T) =CBS 11679(T)) is the type strain of this novel taxon.

Genome Sequence of the Lantibiotic Bacteriocin Producer Streptococcus salivarius Strain K12

Streptococcus salivarius is a prevalent commensal species of the oropharyngeal tract. S. salivarius strain K12 is an isolate from the saliva of a healthy child, used as an oral probiotic. Here, we report its genome sequence, i.e., the full sequence of the 190-kb megaplasmid pSsal-K12 and a high-quality draft 2.2-Gb chromosomal sequence.

Sequence analysis of plasmid pSP02 from Bifidobacterium longum M62 and construction of pSP02-derived cloning vectors

Replicons from bifidobacteria species are required for the construction of general- and special-purpose vectors that would allow the undertaking of molecular studies of these bacteria. In this work, pSP02, a cryptic plasmid from Bifidobacterium longum M62, was cloned, sequenced and characterized. pSP02 was found to consist of 4896bp with four ORFs coding for proteins over 50 amino acids long. Among the deduced protein sequences only a replicase (RepA) and a mobilization-like protein (MobA) showed known functional domains. Similar to previously described bifidobacterial plasmids, the organization of the putative ori region of pSP02 resembles that of the theta-replicating plasmids of Gram-positives. In spite of this, hybridization experiments detected single stranded (ss)-DNA as an intermediate product in the pSP02 replication, demonstrating it follows the rolling-circle (RC) replication mode. The ori region of pSP02 was used to construct a series of first generation cloning vectors able to replicate in many bifidobacterial species. Real time quantitative PCR established the copy number of pSP02 and its derived vectors to be around 12 copies per chromosome equivalent. pSP02-derivatives showed full segregational and structural stability even in the absence of antibiotic selection.

Antibiotic resistance-susceptibility profiles of Lactobacillus strains from Lighvan, a traditional Iranian raw milk cheese

This research was supported by a grant from the Natural Resources Faculty of Tehran University.The genus Capoeta in Iran is highly diversified with 14 species and is one of the most important freshwater fauna components of the country. Central Iran is a region with high number of endemism in other freshwater fish species, though the present species was recognized as C. aculeata (Valenciennes, 1844), widely distributed within Kavir and Namak basins. However previous phylogenetic and phylogeographic studies found that populations of Nam River, a tributary of the Hableh River in central Iran are different from the other species. In this study, the mentioned population is described as a new species based on morphologic and genetic characters.This research was founded by projects from the Vice Chancellor for Research, University of Tehran, lran (Grant no. 61 22-TNS2007), and the Spanish Ministry of Science and lnnovation (Reference, AGL2007-61869-ALI).