The person-to-person transmission landscape of the gut and oral microbiomes.

The human microbiome is an integral component of the human body and a co-determinant of several health conditions1,2. However, the extent to which interpersonal relations shape the individual genetic makeup of the microbiome and its transmission within and across populations remains largely unknown3,4. Here, capitalizing on more than 9,700 human metagenomes and computational strain-level profiling, we detected extensive bacterial strain sharing across individuals (more than 10 million instances) with distinct mother-to-infant, intra-household and intra-population transmission patterns. Mother-to-infant gut microbiome transmission was considerable and stable during infancy (around 50% of the same strains among shared species (strain-sharing rate)) and remained detectable at older ages. By contrast, the transmission of the oral microbiome occurred largely horizontally and was enhanced by the duration of cohabitation. There was substantial strain sharing among cohabiting individuals, with 12% and 32% median strain-sharing rates for the gut and oral microbiomes, and time since cohabitation affected strain sharing more than age or genetics did. Bacterial strain sharing additionally recapitulated host population structures better than species-level profiles did. Finally, distinct taxa appeared as efficient spreaders across transmission modes and were associated with different predicted bacterial phenotypes linked with out-of-host survival capabilities. The extent of microorganism transmission that we describe underscores its relevance in human microbiome studies5, especially those on non-infectious, microbiome-associated diseases.

Metaprofiling of the bacterial community in sorghum silages inoculated with lactic acid bacteria.

AIMS: To characterize the fermentation process and bacterial diversity of sorghum silage inoculated with Lactiplantibacillus plantarum LpAv, Pediococcus pentosaceus PpM and Lacticaseibacillus paracasei LcAv. METHODS AND RESULTS: Chopped sorghum was ensiled using the selected strains. Physicochemical parameters (Ammonia Nitrogen/Total Nitrogen, Dry Matter, Crude Protein, Acid Detergent Fibre, Neutral Detergent Fibre, Acid Detergent Lignin, Ether Extract and Ashes), bacterial counts, cell cytometry and 16sRNA sequencing were performed to characterize the ensiling process and an animal trial (BALB/c mice) was conducted in order to preliminary explore the potential of sorghum silage to promote animal gut health. After 30 days of ensiling, the genus Lactobacillus comprised 68.4 ± 2.3% and 73.5 ± 1.8% of relative abundance, in control and inoculated silages respectively. Richness (Chao1 index) in inoculated samples, but not in control silages, diminished along ensiling, suggesting the domination of fermentation by the inoculated LAB. A trend in conferring enhanced protection against Salmonella infection was observed in the mouse model used to explore the potential to promote gut health of sorghum silage. CONCLUSIONS: The LAB strains used in this study were able to dominate sorghum fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report using metaprofiling of 16sRNA to characterize sorghum silage, showing a microbiological insight where resident and inoculated LAB strains overwhelmed the epiphytic microbiota, inhibiting potential pathogens of the genus Klebsiella.

[Atypical spoilage microorganisms in Argentinean yogurts: Gas-producing molds and bacteria of the genus Gluconobacter]. / Alterantes microbianos atípicos en yogures argentinos: mohos gasógenos y bacterias del género Gluconobacter.

Microbial food alterations lead to unfit products for consumption, and their discarding, to significant economic losses for the food industry. During storage, fresh foods offer available niches for the survival and growth of undesirable microorganisms. In dairy products, data regarding spoilage and/or pathogenic bacteria is better documented than those for molds and yeasts. Dairy products are less susceptible to mold's contamination than products such as fruits and vegetables, due to their refrigerated storage; their elaboration from heat-treated milk and, for fermented ones, the dominant microbiota that acidifies the medium. However, even cheeses and yogurts may be susceptible to mold contamination. Atypical cases of yogurt samples containing spoilage microorganisms not previously reported (molds producing gas and bacteria of the genus Gluconobacter) in Argentinean fermented milks are presented here. For yogurt, in particular, the "classic" altering organisms were always being yeasts, and in other countries, molds belonging to the genus Aspergillus.

Lactobacillus fermentum: Could EPS production ability be responsible for functional properties?

Exopolysaccharides (EPS) production is a characteristic that has been widely described for many lactic acid bacteria (LAB) of different genera and species, but little is known about the relationship between the functional properties of the producing bacteria and EPS synthesis. Although many studies were addressed towards the application of EPS-producing LAB in the manufacture of several dairy products (fermented milk, cheese) due to their interesting technological properties (increased hardness, water holding capacity, viscosity, etc.), there are not many reports about the functional properties of the EPS extract itself, especially for the genus Lactobacillus. The aim of the present revision is to focus on the species Lactobacillus fermentum with reported functional properties, with particular emphasis on those strains capable of producing EPS, and try to establish if there is any linkage between this property and their functional/probiotic roles, considering the most recent bibliography.

Exopolysaccharide from Lactobacillus fermentum Lf2 and its functional characterization as a yogurt additive.

Lactobacillus fermentum Lf2 is a strain which is able to produce high levels (approximately 1 g/l) of crude exopolysaccharide (EPS) when it is grown in optimised conditions. The aim of this work was to characterize the functional aspects of this EPS extract, focusing on its application as a dairy food additive. Our findings are consistent with an EPS extract that acts as moderate immunomodulator, modifying s-IgA and IL-6 levels in the small intestine when added to yogurt and milk, respectively. Furthermore, this EPS extract, in a dose feasible to use as a food additive, provides protection against Salmonella infection in a murine model, thus representing a mode of action to elicit positive health benefits. Besides, it contributes to the rheological characteristics of yogurt, and could function as a food additive with both technological and functional roles, making possible the production of a new functional yogurt with improved texture.

Role of Probiotics, Prebiotics, and Synbiotics in the Elderly: Insights Into Their Applications.

Elderly people are an important part of the global population who suffer from the natural processes of senescence, which lead to changes in the gut microbiota composition. These modifications have a great impact on their quality of life, bringing a general putrefactive and inflammatory status as a consequence. Some of the most frequent conditions related to this status are constipation, undernutrition, neurodegenerative diseases, susceptibility to opportunistic pathogens, and metabolic disbalance, among others. For these reasons, there is an increasing interest in improving their quality of life by non-invasive treatments such as the consumption of probiotics, prebiotics, and synbiotics. The aim of the present mini-review is to describe the benefits of these functional supplements/food according to the most recent clinical and pre-clinical studies published during the last decade. In addition, insights into several aspects we consider relevant to improve the quality of future studies are provided.

Structural characterisation of two medium molecular mass exopolysaccharides produced by the bacterium Lactobacillus fermentum Lf2.

Under optimized conditions, the lactic acid bacterium Lactobacillus fermentum Lf2 secretes up to 2 gL-1 of a mixture of polysaccharides into the fermentation medium when grown on sucrose. Earlier studies had shown that the mixture is biologically active and work was undertaken to characterise the polysaccharides. Preparative size exclusion chromatography was used to separate a high molecular mass ß-glucan (weight average mass of 1.23 × 106 gmol-1) from two medium molecular mass polysaccharides (weight average mass of 8.8 × 104 gmol-1). Under optimized growth conditions, the medium molecular mass polysaccharides accounted for more than 75% of the mixture by weight. Monomer, linkage analysis and NMR spectroscopy of the medium molecular mass polysaccharides, and material isolated after their Smith degradation, was used to identify the structure of the component polysaccharides. The mixture contains two novel polysaccharides. The first has a main chain of ß-1,6-linked galactofuranoses which is non-stoichiometrically 2-O-glucosylated. The degree of substitution at the 2-position, with α-D-Glcp, depends on the fermentation conditions; under optimized conditions greater than 80% 2-O-α-D-glucosylation was observed. The second polysaccharide is a heteroglycan with four monosaccharides in the repeat unit: residual signals in the NMR suggest that the sample also contains trace amounts (<3%) of cell wall polysaccharides.

Isolation and characterization of a high molecular mass ß-glucan from Lactobacillus fermentum Lf2 and evaluation of its immunomodulatory activity.

When grown in a semi-defined medium, L. fermentum Lf2 synthesizes significant quantities (∼2 g/L) of two exopolysaccharides (EPS). The two EPS were separated by preparative size exclusion chromatography to give a high molecular mass ß-glucan (1.23 × 106 Da) and a medium molecular mass heteroglycan (8.8 × 104 Da). The structure of the high molecular mass ß-glucan was determined using a combination of NMR spectroscopy, monomer and linkage analysis. The EPS has the following structure: The immunomodulatory activity of the high molecular mass EPS was studied in peripheral blood mononuclear cells (PBMC). Exposure of PBMC to an aqueous solution of the EPS for 24 h led to increased cell proliferation, changes in expression of the cytokines CD14 and TLR2, and to an increase in production of TNF-α compared to controls. In contrast, when cells that had been treated with EPS for 24 h and from which the EPS had been removed, were subsequently exposed to the bacterial antigen LPS very low levels of TNF-α production were observed. This result indicates that the EPS imparts immunotolerance in PBMC. An ability to modulate the release of the proinflammatory mediators, such as TNF-α, is an important goal in the development of therapies for the treatment of diseases, such as Crohn's disease and ulcerative colitis, associated with excessive release of inflammatory mediators.

PCR method for detection and identification of Lactobacillus casei/paracasei bacteriophages in dairy products.

Bacteriophage infections of starter lactic acid bacteria (LAB) pose a serious risk to the dairy industry. Nowadays, the expanding use of valuable Lactobacillus strains as probiotic starters determines an increase in the frequency of specific bacteriophage infections in dairy plants. This work describes a simple and rapid Polymerase Chain Reaction (PCR) method that detects and identifies bacteriophages infecting Lactobacillus casei/paracasei, the main bacterial species used as probiotic. Based on a highly conserved region of the NTP-binding genes belonging to the replication module of L. casei phages phiA2 and phiAT3 (the only two whose genomes are completely sequenced), a pair of primers was designed to generate a specific fragment. Furthermore, this PCR detection method proved to be a useful tool for monitoring and identifying L. casei/paracasei phages in industrial samples since specific PCR signals were obtained from phage contaminated milk (detection limit: 10(4) PFU/mL milk) and other commercial samples (fermented milks and cheese whey) that include L. casei/paracasei as probiotic starter (detection limit: 10(6) PFU/mL fermented milk). Since this method can detect the above phages in industrial samples and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms, or processes which involve phage-deactivating conditions.

Draft Genome Sequence of Lactobacillus fermentum Lf2, an Exopolysaccharide-Producing Strain Isolated from Argentine Cheese.

Lactobacillus fermentum Lf2, an Argentine cheese isolate, can produce high concentrations of exopolysaccharides (EPS). These EPS were shown to improve the texture and rheology of yogurt, as well as to play a protective role in mice exposed to Salmonella enterica serovar Typhimurium. Three gene clusters potentially involved in EPS production were identified in different locations of the L. fermentum Lf2 genome.

Characterization of spontaneous phage-resistant derivatives of Lactobacillus delbrueckii commercial strains.

A total of 44 spontaneous phage-resistant mutants were isolated from three commercial Lactobacillus delbrueckii strains by secondary culture and agar plate methods. Phenotypic characteristics related to their phage-resistance capacities, i.e. plaquing efficiency, phage-resistance stability, lysogeny and adsorption rates were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying and proteolytic activities and acidification kinetics) properties of mutants were also studied. Amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) was applied to confirm strain identity at the subspecies level. Random amplification of polymorphic DNA (RAPD-PCR) was used to determine genetic diversity among the isolates and their respective parent strains. The secondary culture method was the most useful for obtaining phage-resistant mutants. Phage resistance stability was a variable property among the isolates, but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of spontaneous lysogeny was demonstrated. Adsorption rates were heterogeneously distributed among the three groups of mutants. All mutants isolated from two sensitive strains were similar to them with respect to technological properties. Two groups of mutants with distinctive technological properties were isolated from the other sensitive strain. PCR-ARDRA revealed that two out of three sensitive strains identified commercially as Lb. delbrueckii subsp. bulgaricus were actually Lb. delbrueckii subsp. lactis. Some of the phage-resistant mutants that were obtained might be used in culture rotation programs without regulatory restrictions when commercial strains become sensitive to phages present in industrial environments.

Alterantes microbianos atípicos en yogures argentinos: mohos gasógenos y bacterias del género Gluconobacter / Atypical spoilage microorganisms in Argentinean yogurts: Gas-producing molds and bacteria of the genus Gluconobacter

RESUMEN El deterioro microbiológico de alimentos conduce a productos no aptos para consumo, y su descarte, a importantes pérdidas económicas para la industria alimenticia. Durante su almacenamiento, los alimentos frescos representan nichos atractivos para la supervivencia y el crecimiento de microorganismos indeseables. En productos lácteos, la presencia de alterantes o patógenos bacterianos está mejor documentada que la de mohos y levaduras. Estos productos son menos proclives al deterioro por mohos que otros, como frutas y verduras, debido a su almacenamiento refrigerado, su elaboración a partir de leche tratada térmicamente y, para fermentados, a la microbiota dominante, que acidifica el medio. Sin embargo, incluso quesos y yogures pueden sufrir deterioro por mohos. Este trabajo presenta casos atípicos de muestras de yogur con desarrollo de mohos gasógenos y bacterias del géneroGluconobactercomo microorganismos alterantes no reportados previamente como tales en leches fermentadas argentinas. Los organismos alterantes «clásicos¼ de yogur fueron siempre levaduras y, en otros países, mohos del géneroAspergillus.

ABSTRACT Microbial food alterations lead to unfit products for consumption, and their discarding, to significant economic losses for the food industry. During storage, fresh foods offer available niches for the survival and growth of undesirable microorganisms. In dairy products, data regarding spoilage and/or pathogenic bacteria is better documented than those for molds and yeasts. Dairy products are less susceptible to mold's contamination than products such as fruits and vegetables, due to their refrigerated storage; their elaboration from heat-treated milk and, for fermented ones, the dominant microbiota that acidifies the medium. However, even cheeses and yogurts may be susceptible to mold contamination. Atypical cases of yogurt samples containing spoilage microorganisms not previously reported (molds producing gas and bacteria of the genusGluconobacter) in Argentinean fermented milks are presented here. For yogurt, in particular, the "classic" altering organisms were always being yeasts, and in other countries, molds belonging to the genusAspergillus.

Technological, rheological and sensory characterizations of a yogurt containing an exopolysaccharide extract from Lactobacillus fermentum Lf2, a new food additive.

Lactobacillus fermentum Lf2, an autochthonous strain isolated as a non starter culture in Cremoso cheese, produces high EPS levels (~1g/L) in optimized conditions (SDM broth, pH6.0, 30°C, 72h). Technological (texture profile and rheological analysis) and sensory properties of non-fat yogurts with 300 and 600mg EPS/L were studied at 3 and 25days after manufacture. Yogurts with different EPS concentrations showed higher hardness values than the control group at both periods of time, being the only significant difference that remained stable during time. The consistency index was also higher for the treated samples at both times evaluated, being significantly different for samples with 300mg/L of EPS extract, while the flow behavior index was lower for EPS-added yogurts. The thixotropic index was lower (P<0.05) for samples with the highest EPS extract concentration at the end of the storage time. Regarding the sensory analysis, those yogurts with 600mg/L of EPS extract presented the highest values of consistency at 3days of storage. No considerable differences for defects (milk powder, acid, bitter and cooked milk flavors) were perceived between treated and control samples at both times evaluated. Syneresis was also studied and samples with 600mg/L of EPS extract presented the lowest syneresis values at 25days of storage, which considerably decreased with the time of storage. In conclusion, the EPS from L. fermentum Lf2, used as an additive, provided yogurt with creamy consistency and increased hardness, without the presence of unwanted defects and improving the water holding capacity of the product. All the analysis done showed the potential of this extract to be used as a technofunctional natural ingredient, and it should be considered its positive impact on health, according to previous studies.

Genome analysis of two virulent Streptococcus thermophilus phages isolated in Argentina.

Two Streptococcus thermophilus phages (ALQ13.2 and phiAbc2) were previously isolated from breakdowns of cheese manufacture in Argentina. Complete nucleotide sequence analysis indicated that both phages contained linear double-stranded DNA: 35,525 bp in length for the pac-type phage ALQ13.2 and 34,882 bp for the cos-type phage phiAbc2. Forty-four and 48 open reading frames (ORF) were identified for ALQ13.2 and phiAbc2, respectively. Comparative genomic analysis showed that these isolates shared many similarities with the eight previously studied cos- and pac-phages infecting different S. thermophilus strains. In particular, part of the phiAbc2 genome was highly similar to a region of phage 7201, which was thought to be unique to this latter phage. Protein analysis of the pac-phage ALQ13.2 using SDS polyacrylamide gel electrophoresis (SDS-PAGE) identified three major proteins and seven minor proteins. Parallel structural proteome analysis of phiAbc2 revealed seven protein bands, two of which were related to major structural proteins, as expected for a cos-type phage. Similarities to other S. thermophilus phages suggest that the streptococcal phage diversity is not extensive in worldwide dairy factories possibly because related high-performing bacterial strains are used in starter cultures.

Detection and characterization of Streptococcus thermophilus bacteriophages by use of the antireceptor gene sequence.

In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 10(5) PFU ml(-1)).