Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains.

The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.

Recommended minimal standards for description of new taxa of the genera Bifidobacterium, Lactobacillus and related genera.

Minimal standards for the description of new cultivable strains that represent novel genera and species belonging to the genera Bifidobacterium, Lactobacillus and related genera are proposed in accordance with Recommendation 30b of the Bacteriological Code (1990 Revision): the description of novel species should be based on phenotypic, genotypic and ecological characteristics to ensure a rich polyphasic characterization. Concerning genotypic characterization, in addition to DNA G+C content (mol%) data, the description should be based on DNA-DNA hybridization (DDH), 16S rRNA gene sequence similarities and at least two housekeeping gene (e.g. hsp60 and recA) sequence similarities. DDH might not be needed if the 16S rRNA gene sequence similarity to the closest known species is lower than 97 %. This proposal has been endorsed by members of the Subcommittee on the Taxonomy of Bifidobacterium, Lactobacillus and related organisms of the International Committee on the Systematics of Prokaryotes.

Amarone: a modern wine coming from an ancient production technology.

Amarone wine is a renowned dry red wine produced in Valpolicella (Verona, Northern Italy). It is made from local grapes varieties (Corvina, Rondinella, and Molinara) that are slowly dried under natural conditions during the fall into winter. After the postharvest drying, carried out for several weeks in dedicated lofts called fruttaio, the grapes are vinified: crushed, given prefermentative cold maceration, undergo alcoholic fermentation on the skins, malolactic fermentation, and finally maturation. The partially dried grapes are traditionally crushed during the second half of January to February. Because cellar conditions are unfavorable for either alcohol or malolactic fermentation, selected microbial cultures (yeasts and malolactic bacteria) are often necessary to correctly manage fermentation. The progress of both fermentation processes needs constant surveillance. During maturation conducted in vessels or wooden containers (tonneau in durmast oak), clarification and stabilization lead to improvements in quality. Product specifications require that Amarone not be bottled before the wine has been aged for 2years (Anonymous (2010). Disciplinare di produzione dei vini a denominazione di Origine Controllata e Garantita "Amarone della Valpolicella". Gazzetta Ufficiale della Repubblica Italiana. Serie generale n. 84. April 12). Amarone achieved its DOCG (Controlled and Guaranteed Denomination) status in 2010.

Reclassification of Lactobacillus catenaformis (Eggerth 1935) Moore and Holdeman 1970 and Lactobacillus vitulinus Sharpe et al. 1973 as Eggerthia catenaformis gen. nov., comb. nov. and Kandleria vitulina gen. nov., comb. nov., respectively.

The development of molecular tools and in particular the use of 16S rRNA gene sequencing has had a profound effect on the taxonomy of many bacterial groups. Gram-positive organisms that encompass the genera Lactobacillus and Clostridium within the Firmicutes are examples of taxa that have undergone major revisions based on phylogenetic information. A consequence of these reorganizations is that a number of organisms are now recognized as being misclassified. Previous studies have demonstrated that Lactobacillus catenaformis and Lactobacillus vitulinus are phylogenetically unrelated to Lactobacillus sensu stricto, being placed within the Clostridia rRNA cluster XVII. Based on the phenotypic, chemotaxonomic and phylogenetic data presented, it is proposed that L. catenaformis and L. vitulinus be reclassified in two new genera, named respectively Eggerthia gen. nov., with the type species Eggerthia catenaformis gen. nov., comb. nov. (type strain DSM 20559(T) = ATCC 25536(T) = CCUG 48174(T) = CIP 104817(T) = JCM 1121(T)) and Kandleria gen. nov., with the type species Kandleria vitulina gen. nov., comb. nov. (type strain LMG 18931(T) = ATCC 27783(T) = CCUG 32236(T) = DSM 20405(T) = JCM 1143(T)).

A novel bioengineering clinical device testing cellular homeostatic potentials to customize and monitor nutritional-related age-management interventional strategies.

Most devices assessing body composition harbor a number of drawbacks and hardly assess the phenomena taking place at a cellular membrane level. The present single-frequency bioelectrical potential homeostatic structure analysis (PHoSA) technology requires only a proper hands contact on fixed electrodes and determines the phase displacement between tested current and voltage by using a 50-KHz alternate sinusoidal current. This allows quick testing time with high degree of precision, sensitivity, and specificity of sectorial functional body compartments analysis. Such assessment may prove to be an integrated part of either a diagnostic workup or monitoring tool in tailoring nutritional/nutraceutical, pharmacological, and exercise activity, all being framed within a proactive, preventive, age-intervention management strategy.

Molecular diversity and transferability of the tetracycline resistance gene tet(M), carried on Tn916-1545 family transposons, in enterococci from a total food chain.

In the present study, 20 enterococci belonging to the species Enterococcus faecalis (12 strains), Enterococcus faecium (4), Enterococcus durans (2), Enterococcus hirae (1) and Enterococcus mundtii (1) and originating from a total production chain of swine meat commodities were analysed to investigate the diversity of their tetracycline resistance gene tet(M). PCR-RFLP and sequence analysis showed that the tet(M) gene of most strains can be correlated with the Tn916 transposon. Conversely, tet(M) of six E. faecalis and the E. hirae strain, all isolated from pig faecal samples, may be associated with previously undescribed members of the Tn916-1545 transposon family. In vitro filter conjugation trials showed the ability of 50% of the enterococcal strains, including E. mundtii, to transfer the tet(M) gene (and the associated Tn916 and new transposons) to E. faecalis or Listeria innocua recipient strains. tet(M) gene transfer to L. innocua recipient was also directly observed in meat food products. Collectively, these sequence and conjugation data indicate that various transposons can be responsible of the spread of tetracycline resistance in enterococci and validate the opinion that Enterococcus species are important sources of antibiotic resistance genes for potentially pathogenic bacteria occurring in the food chain.

Characterization of tetracycline-resistant Streptococcus thermophilus isolates from Italian soft cheeses.

Tetracycline-resistant Streptococcus thermophilus isolates from soft cheeses harbored the genes tet(S), tet(M), and tet(L). Molecular analysis of these genes revealed their expression, localization on plasmids or Tn916-Tn1545 family transposons, and their similarity with published sequences. The study highlights the importance of an accurate safety assessment of using S. thermophilus as a starter culture.

Study of microbial diversity in raw milk and fresh curd used for Fontina cheese production by culture-independent methods.

The bacterial populations of raw milk employed for the production of Fontina cheese in alpine farms located in different valleys and altitudes (from 700 to 2246 m above sea level) were investigated by culture independent techniques. Total microbial DNA was isolated from milk and curd samples and used as template in Polymerase Chain Reaction (PCR) to study the hypervariable V3 region of the bacterial 16S rRNA gene and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE). Representative bands of DGGE patterns were sequenced for identification purposes. The use of universal primer for PCR-DGGE allowed the description of the bacterial community, not only for the presence of lactic acid bacteria, but also for other adventitious species. DGGE profiles obtained from milk and fresh curd samples were generally different and typical for each farm, although some recurrent bands were observed. Cluster analysis of DGGE profiles did not show high similarity among samples and it was probably dependent on the different geographical areas of pastures. Some Lactic Acid Bacteria (LAB) recurred in many samples (Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Lactococcus lactis, Leuconostoc lactis) indicating that alpine milk is a preferential niche for their colonization. The microbiota included not only mesophilic and thermoresistant LAB but also adventitious bacteria (Macrococcus caseolyticus, Rothia spp.) and psychrotrophic bacteria (Chryseobacterium spp., Pseudomonas spp.), that were found in almost all samples, but disappeared after the warming up at 47-48 degrees C of coagulated milk. Pantoea spp. was primarily found in curds and only with a low incidence in milk samples, indicating the environmental origin. Finally the sequencing data confirmed the presence of E. faecium, E. faecalis and S. thermophilus as major species present in the curd. These species were found also in raw milk, proving its importance as source of the typical fermenting microflora.

Genetic modification of Lactobacillus plantarum by heterologous gene integration in a not functional region of the chromosome.

This report describes the vector-free engineering of Lactobacillus plantarum by chromosomal integration of an exogenous gene without inactivation of physiological traits. The integrative plasmid vector pP7B6 was derived from pGIP73 by replacing the cbh site, encoding the L. plantarum conjugated bile salt hydrolase, with the prophage fragment P7B6, from L. plantarum Lp80 (DSM 4229). Plasmid pP7B6NI was obtained by inserting the nisin immunity gene nisI of Lactococcus lactis subsp. lactis DSM 20729, preceded by the constitutive promoter P32 from the same strain, in a unique XbaI site of fragment P7B6 and was used to electrotransform L. plantarum Lp80. A food grade recombinant L. plantarum Lp80NI, with 480-fold higher immunity to nisin than the wild type, was derived by integration of pP7B6NI followed by the excision of pP7B6. Polymerase chain reaction tests demonstrated that the integration of nisI in the prophage region had occurred and that the erythromycin resistance marker from pP7B6 was lost. Fifteen among 31 L. plantarum strains tested hybridized with P7B6, indicating that the integration of pP7B6-derived vectors might occur in some other L. plantarum strains. This was experimentally confirmed by constructing the recombinant strain L. plantarum LZNI from the dairy isolate L. plantarum LZ (LMG 24600).

Antibiotic resistance genes and identification of staphylococci collected from the production chain of swine meat commodities.

Staphylococci harbouring antibiotic resistance (AR) genes may represent a hazard for human health and, as other resistant food-related bacteria, they contribute to the spread of AR. In this study, we isolated resistant staphylococci from an entire swine production chain and investigated the occurrence of 11 genes [aac(6')Ie-aph(2'')Ia, blaZ, mecA, vanA, vanB, ermA, ermB, ermC, tet(M), tet(O) and tet(K)] encoding resistance to some antibiotics largely used in clinical practice. The 66 resistant staphylococcal isolates were identified as Staphylococcus epidermidis (27 isolates), Staphylococcus aureus (12), Staphylococcus xylosus (12), Staphylococcus simulans (5), Staphylococcus pasteuri (4), Staphylococcus carnosus (3), Staphylococcus lentus (2) and Staphylococcus sciuri (1). Specific-PCR detection of AR genes showed the prevalence of the tet(K) gene in most of the isolates (89.4%), followed by tet(M) and ermC (about 75%); mecA was detected in more than half of S. aureus and S. epidermidis isolates. The genes vanA and vanB were not retrieved. It was found that a high proportion of coagulase-positive and -negative isolates are multidrug-resistant and some of them carry up to six AR genes. Our findings show that the swine production chain is a source of antibiotic-resistant staphylococci suggesting the importance of resistance surveillance in the food production environment.

Multimodal phylogeny for taxonomy: integrating information from nucleotide and amino acid sequences.

The crucial role played by the analysis of microbial diversity in biotechnology-based innovations has increased the interest in the microbial taxonomy research area. Phylogenetic sequence analyses have contributed significantly to the advances in this field, also in the view of the large amount of sequence data collected in recent years. Phylogenetic analyses could be realized on the basis of protein-encoding nucleotide sequences or encoded amino acid molecules: these two mechanisms present different peculiarities, still starting from two alternative representations of the same information. This complementarity could be exploited to achieve a multimodal phylogenetic scheme that is able to integrate gene and protein information in order to realize a single final tree. This aspect has been poorly addressed in the literature. In this paper, we propose to integrate the two phylogenetic analyses using basic schemes derived from the multimodality fusion theory (or multiclassifier systems theory), a well-founded and rigorous branch for which its powerfulness has already been demonstrated in other pattern recognition contexts. The proposed approach could be applied to distance matrix-based phylogenetic techniques (like neighbor joining), resulting in a smart and fast method. The proposed methodology has been tested in a real case involving sequences of some species of lactic acid bacteria. With this dataset, both nucleotide sequence- and amino acid sequence-based phylogenetic analyses present some drawbacks, which are overcome with the multimodal analysis.

Differences in faecal bacterial communities in coeliac and healthy children as detected by PCR and denaturing gradient gel electrophoresis.

Coeliac disease (CD) is a chronic inflammatory disorder of the small intestinal mucosa. Scientific evidence supports a role of the gut microbiota in chronic inflammatory disorders; yet information is not specifically available for CD. In this study, a comparative denaturing gradient gel electrophoresis analysis of faecal samples from coeliac children and age-matched controls was carried out. The diversity of the faecal microbiota was significantly higher in coeliac children than in healthy controls. The presence of the species Lactobacillus curvatus, Leuconostoc mesenteroides and Leuconostoc carnosum was characteristic of coeliac patients, while that of the Lactobacillus casei group was characteristic of healthy controls. The Bifidobacterium population showed a significantly higher species diversity in healthy children than in coeliacs. In healthy children, this population was characterized by the presence of Bifidobacterium adolescentis. Overall, the results highlighted the need for further characterization of the microbiota in coeliac patients, and suggested a potential role of probiotics and/or prebiotics in restoring their gut microbial balance.

On species descriptions based on a single strain: proposal to introduce the status species proponenda (sp. pr.).

A survey of the descriptions of novel bacterial species published in the period 1996-2006 revealed that a large number of taxonomic descriptions are still based on one or a few strains. This situation determines that not only species descriptions, but also proposals to create higher ranks, are actually based on very few strains, which could produce a highly biased scenario. The encouragement to include a reasonable number of strains in species descriptions has been largely disregarded after its proposal, since acceptance of such descriptions relies mainly on editors' and reviewers' opinions. This observation and other considerations lead us to propose the creation of the status species proponenda (sp. pr.), as a compromise between the need for scientific description of biodiversity and exchange of data and the good taxonomic practice of including a sufficient number of strains in descriptions of species and higher taxonomic ranks.

Taxonomy of Lactobacilli and Bifidobacteria.

Genera Lactobacillus and Bifidobacterium include a large number of species and strains exhibiting important properties in an applied context, especially in the area of food and probiotics. An updated list of species belonging to those two genera, their phylogenetic relationships and other relevant taxonomic information are reviewed in this paper. The conventional nature of taxonomy is explained and some basic concepts and terms will be presented for readers not familiar with this important and fast-evolving area, which importance is often underestimated. The analysis of biodiversity and its cataloguing, i.e. taxonomy, constitute the basis for applications and scientific communication: reliable identification and correct naming of bacterial strains are not only primary aims of taxonomic studies, but also fundamental elements in an applied context, for the tracking of probiotic strains and a non fraudulent labelling of fermented milks and pharmaceutical products containing probiotic microorganisms. A number of resources freely available have been listed and their use is suggested for people concerned with different aspects of taxonomy. Some perspectives in taxonomy have been outlined, in particular considering the role of culture independent analyses to reveal the still unknown and uncultured microorganisms. Finally, the impact of the availability of whole-genome sequences in taxonomy is briefly explained: they have already begun to give insights on bacterial evolution, which will surely have implications on taxonomy, even if the analysis of data for lactic acid bacteria is still limited to few species.

Analysis of bifidobacterial evolution using a multilocus approach.

Bifidobacteria represent one of the most numerous groups of bacteria found in the gastrointestinal tract of humans and animals. In man, gastrointestinal bifidobacteria are associated with health effects and for this reason they are often used as functional ingredients in food and pharmaceutical products. Such applications may benefit from or require a clear and reliable bifidobacterial species identification. The increasing number of available bacterial genome sequences has provided a large amount of housekeeping gene sequences that can be used both for identification of bifidobacterial species as well as for understanding bifidobacterial evolution. In order to assess their relative positions in the evolutionary process, fragments from seven conserved genes, clpC, dnaB, dnaG, dnaJ1, purF, rpoC and xfp, were sequenced from each of the currently described type strains of the genus Bifidobacterium. The results demonstrate that the concatenation of these seven gene sequences for phylogenetic purposes allows a significant increase in the discriminatory power between taxa.

Lactobacillus durianis Leisner et al. 2002 is a later heterotypic synonym of Lactobacillus vaccinostercus Kozaki and Okada 1983.

The taxonomic status of the species Lactobacillus durianis and Lactobacillus vaccinostercus is briefly summarized and experimental evidence concerning their similarity is presented. Highly similar 16S rRNA gene sequences (99.8 % similarity over 1,523 bp), partial recA gene sequences (99.5 % similarity over 600 bp) and partial hsp60 gene sequences (99.1 % similarity over 924 bp) suggest that the two species are closely related. Moreover, a high DNA-DNA binding level (87 %) and similar genomic DNA G+C contents (41-44 mol% for both species) as well as similar biochemical characteristics support the evidence that they constitute a single species. Consequently, according to Rules 38 and 42 of the Bacteriological Code, the name Lactobacillus vaccinostercus, the oldest legitimate name, must be maintained and the name Lactobacillus durianis should be considered a later heterotypic synonym.

Lactobacillus paracasei A survives gastrointestinal passage and affects the fecal microbiota of healthy infants.

This study focuses on the potentiality of a putative probiotic strain, Lactobacillus paracasei A, to survive gastrointestinal (GI) passage and modulate the resident microbiota of healthy infants. In a placebo-controlled study, 26 children aged 12-24 months received 100 g/day of either fermented milk containing strain A or pasteurized yogurt for four weeks. Fecal samples were analyzed before starting the administration, after 1, 3 and 4 weeks of consumption and after washout. The fate of strain A was followed by means of a newly developed PCR targeting a strain-specific genomic marker. The composition and dynamics of fecal microbial communities during the study were analyzed by culturing on selective media and by the PCR-denaturing gradient gel electrophoresis (DGGE) technique using universal and group-specific (Lactobacillus and Bifidobacterium) primers. The variation in enzymatic activities in infant feces during probiotic consumption was also analyzed. Strain A survived in fecal samples in most (92%) of the infants examined after 1 week of consumption, and temporarily dominated the intestinal Lactobacillus community. The administration of L. paracasei A led to a significant increment in the Lactobacillus population, while a moderate effect upon the main bacterial groups in the GI ecosystem was observed. Strain A also affected the diversity of the Lactobacillus and Bifidobacterium populations. The fecal bacterial structure of 1 - 2-year-old infants seems to combine neonate and adult-like features. The microbiota of these subjects promptly responded to probiotic consumption, later restoring the endogenous equilibrium.

Evaluation of recA gene as a phylogenetic marker in the classification of dairy propionibacteria.

The aim of this study was to investigate the validity of recA gene as a molecular marker for the reliable discrimination and classification of dairy propionibacteria and the closely related species. Regions of the recA gene, varying in size between 613 and 677 nucleotides, were sequenced for Propionibacterium acidipropionici, P. cyclohexanicum, P. freudenreichii, P. jensenii, P. microaerophilum and P. thoenii using degenerate consensus primers constructed by aligning recA sequences of some actinobacteria. The 16S rRNA encoding genes for the type and reference strains of the species P. acidipropionici, P. jensenii and P. thoenii were also sequenced to remove ambiguous positions present in the current database reports, such to improve the classification scheme of reference. As found for other bacterial species, recA sequences permitted a better distinction among the dairy propionibacteria considered than 16S rRNA gene. However, the topology of phylogenetic trees constructed on the recA gene regions sequenced and their putative translations appeared rather different and less statistically valid than the 16S rRNA gene tree. In addition, the possibility of designing PCR-based identification and detection tests on the new recA sequences was demonstrated by assessing specific amplification protocols for P. cyclohexanicum and P. microaerophilum.

Reclassification of Leuconostoc argentinum as a later synonym of Leuconostoc lactis.

Leuconostoc argentinum, Leuconostoc lactis and ten related strains from Romanian dairy products formed a single cluster, clearly separated from other Leuconostoc species, after numerical analysis of repetitive extragenic palindromic-PCR patterns, whole-cell protein profiles (SDS-PAGE) and fluorescent amplified fragment length polymorphism (FAFLP) band patterns. 16S rRNA gene sequence analysis confirmed a very high similarity between both type strains and representative dairy isolates (>99.6 %). DNA-DNA hybridization experiments revealed high relatedness values between the type strains of L. argentinum and L. lactis and between these strains and representative Romanian strains. These data and the lack of phenotypic distinctive characteristics demonstrate that L. argentinum and L. lactis are synonymous.