Database selection for shotgun metaproteomic of low-diversity dairy microbiomes.

The metaproteomics field has recently gained more and more interest as a valuable tool for studying both the taxonomy and function of microbiomes, including those used in food fermentations. One crucial step in the metaproteomics pipeline is selecting a database to obtain high-quality taxonomical and functional information from microbial communities. One of the best strategies described for building protein databases is using sample-specific or study-specific protein databases obtained from metagenomic sequencing. While this is true for high-diversity microbiomes (such as gut and soil), there is still a lack of validation for different database construction strategies in low-diversity microbiomes, such as those found in fermented dairy products where starter cultures containing few species are used. In this study, we assessed the performance of various database construction strategies applied to metaproteomics on two low-diversity microbiomes obtained from cheese production using commercial starter cultures and analyzed by LC-MS/MS. Substantial differences were detected between the strategies, and the best performance in terms of the number of peptides and proteins identified from the spectra was achieved by metagenomic-derived databases. However, extensive databases constructed from a high number of available online genomes obtained a similar taxonomical and functional annotation of the metaproteome compared to the metagenomic-derived databases. Our results indicate that, in the case of low-diversity dairy microbiomes, the use of publically available genomes to construct protein databases can be considered as an alternative to metagenome-derived databases.

Genomic and proteomic characterization of vB_SauM-UFV_DC4, a novel Staphylococcus jumbo phage.

Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: • vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. • vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. • vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.

Composition and fate of heat-resistant anaerobic spore-formers in the milk powder production line.

Anaerobic spore-forming bacteria are a continuous threat to the dairy industry due to their ability to withstand processing conditions, such as those during heat treatment. These ubiquitous microorganisms have ample opportunity for multiple entry points into the milk chain, creating food quality and safety issues. Certain spore-formers, namely bacilli and clostridia, are more problematic due to their ability to spoil dairy products and pathogenicity. In this study, we investigated how milk treatment and milk powder production influenced the composition and survival of anaerobic spore-formers. Samples were obtained on three different days (replicate blocks) during the production of dairy powders and examined in a culture-dependent manner using the most probable number method coupled with 16S rRNA amplicon sequencing and metagenomic analysis of the enriched samples. Results revealed that the milk separation greatly affected the spore-former presence and composition which were detected along the entire production line from raw material to milk powders. Throughout the various points of the production line, the occurrence of species belonging to the Bacillus cereus sensu lato was higher than that of clostridia. Sequence variants (SVs) belonging to the anaerobic spore-forming genus Clostridium were taxonomically assigned to two SVs groups and were detected in all three replicate blocks. A total of 19 metagenome-assembled genomes were recovered from nine enrichments. Four near-complete and two medium-quality genomes were found in raw milk/milk powder samples and further assigned as Clostridium tyrobutyricum and Clostridium diolis, which may constitute a problem in the finished dairy product. In conclusion, our findings highlight spore-formers' importance on dairy quality and may aid in their intervention and control in the dairy production line.

Metataxonomic analysis and host proteome response in dairy cows with high and low somatic cell count: a quarter level investigation.

Host response to invasive microbes in the bovine udder has an important role on the animal health and is essential to the dairy industry to ensure production of high-quality milk and reduce the mastitis incidence. To better understand the biology behind these host-microbiome interactions, we investigated the somatic cell proteomes at quarter level for four cows (collected before and after milking) using a shotgun proteomics approach. Simultaneously, we identified the quarter microbiota by amplicon sequencing to detect presence of mastitis pathogens or other commensal taxa. In total, 32 quarter milk samples were analyzed divided in two groups depending on the somatic cell count (SCC). The high SCC group (>100,000 cell/mL) included 10 samples and significant different proteome profiles were detected. Differential abundance analysis uncovers a specific expression pattern in high SCC samples revealing pathways involved in immune responses such as inflammation, activation of the complement system, migration of immune cells, and tight junctions. Interestingly, different proteome profiles were also identified in quarter samples containing one of the two mastitis pathogens, Staphylococcus aureus and Streptococcus uberis, indicating a different response of the host depending on the pathogen. Weighted correlation network analysis identified three modules of co-expressed proteins which were correlated with the SCC in the quarters. These modules contained proteins assigned to different aspects of the immune response, but also amino sugar and nucleotide sugar metabolism, and biosynthesis of amino acids. The results of this study provide deeper insights on how the proteome expression changes at quarter level in naturally infected cows and pinpoint potential interactions and important biological functions during host-microbe interaction.

Lactobacillus gasseri LG-G12 Restores Gut Microbiota and Intestinal Health in Obesity Mice on Ceftriaxone Therapy.

Gut microbiota imbalance is associated with the occurrence of metabolic diseases such as obesity. Thus, its modulation is a promising strategy to restore gut microbiota and improve intestinal health in the obese. This paper examines the role of probiotics, antimicrobials, and diet in modulating gut microbiota and improving intestinal health. Accordingly, obesity was induced in C57BL/6J mice, after which they were redistributed and fed with an obesogenic diet (intervention A) or standard AIN-93 diet (intervention B). Concomitantly, all the groups underwent a treatment phase with Lactobacillus gasseri LG-G12, ceftriaxone, or ceftriaxone followed by L. gasseri LG-G12. At the end of the experimental period, the following analysis was conducted: metataxonomic analysis, functional profiling of gut microbiota, intestinal permeability, and caecal concentration of short-chain fatty acids. High-fat diet impaired bacterial diversity/richness, which was counteracted in association with L. gasseri LG-G12 and the AIN-93 diet. Additionally, SCFA-producing bacteria were negatively correlated with high intestinal permeability parameters, which was further confirmed via functional profile prediction of the gut microbiota. A novel perspective on anti-obesity probiotics is presented by these findings based on the improvement of intestinal health irrespective of undergoing antimicrobial therapy or not.

Synbiotic modulates intestinal microbiota metabolic pathways and inhibits DMH-induced colon tumorigenesis through c-myc and PCNA suppression.

The use of probiotic and synbiotic is a promising strategy to modulate the intestinal microbiota, and thereby modify the risk of diseases. In this study, the effect of probiotic VSL#3, isolated or associated with a yacon-based product (PBY), on the functional metabolic pathways of the microbiota, in a colorectal carcinogenesis model, was evaluated. For this, mice induced to carcinogenesis were fed with standard diet AIN-93 M (CON), diet AIN-93 M and VSL#3 (PRO) or diet AIN-93 M with yacon and VSL#3 (SYN). The SYN group showed a highly differentiated intestinal community based on the MetaCyc pathways. Of the 351 predicted functional pathways, 222 differed between groups. Most of them were enriched in the SYN group, namely: amino acid biosynthesis pathways, small molecule biosynthesis pathways (cofactors, prosthetic groups, electron carriers and vitamins) carbohydrate degradation pathways and fermentation pathways. In addition, the synbiotic was able to stimulate the anti-inflammatory immune response and reduce the gene expression of PCNA and c-myc. Thus, we conclude that the synbiotic impacted more significantly the metabolic functions of the microbiota compared to the isolated use of probiotic. We believe that the enrichment of these pathways can exert antiproliferative action, reducing colorectal carcinogenesis. The prediction of the functional activity of the microbiota is a promising tool for understanding the influence of the microbiome on tumor development.

Longitudinal dynamics of the bovine udder microbiota.

BACKGROUND: In recent years, the number of studies concerning microbiota of the intramammary environment has increased rapidly due to the development of high-throughput sequencing technologies that allow mapping of microbiota without culturing. This has revealed that an environment previously thought to be sterile in fact harbours a microbial community. Since this discovery, many studies have investigated the microbiota of different parts of the udder in various conditions. However, few studies have followed the changes that occur in the udder microbiota over time. In this study, the temporal dynamics of the udder microbiota of 10 cows, five with a low somatic cell count (SCC, SCC < 100,000 cells/mL) and five with a high SCC (SCC > 100,000 cells/mL), were followed over 5 months to gather insights into this knowledge gap. RESULTS: Analysis of the temporal changes in the microbial composition of milk from udders with a low SCC revealed a dynamic and diverse microbiota. When an imbalance due to one dominating genus was recorded, the dominant genus quickly vanished, and the high diversity was restored. The genera dominating in the samples with a high SCC remained the dominant genera throughout the whole sampling period. These cows generally displayed a heightened SCC or an intramammary infection in at least one quarter though-out the sampling period. CONCLUSION: Our results show that the bovine udder has a diverse microbiota, and that the composition and diversity of this community affects udder health with regards to SCC. Understanding what influences the composition and stability of this community has important implications for the understanding, control, and treatment of mastitis.

Chia (Salvia hispanica L.) Flour and Oil Ameliorate Metabolic Disorders in the Liver of Rats Fed a High-Fat and High Fructose Diet.

We hypothesized that the consumption of chia (Salvia hispanica L.) flour (CF) and chia oil (CO) improves metabolic disorders in the liver of Wistar rats (Rattus norvegicus domestica) fed a high-fat and high-fructose (HFHF) diet. The animals were fed a HFHF diet (n = 30) or AIN93-M standard diet (n = 10) for eight weeks. After this period, the animals fed HFHF were divided into three groups (n = 10): HFHF diet, HFHF plus 14.7% of CF, and HFHF plus 4% of CO. Histological and biochemical analyses, gene expression, protein levels related to inflammation, and oxidative stress were evaluated in the liver. The HFHF diet caused lipogenesis, liver steatosis, oxidative stress, and inflammation in the animals. The CF and CO intake increased the liver total antioxidant capacity and superoxide dismutase, decreased nitric oxide levels and liver steatosis. Furthermore, the CF and CO led to the upregulation of Cpt1a and Adipor2, respectively, whereas CF downregulated Srebf1. CO intake decreased blood glucose, triglycerides, and the animals' body weight. Chia did not show effects on mitigating liver pro-inflammatory status, which it may indicate occurs later. The addition of chia into an unbalanced diet is a good and relevant strategy to reduce liver metabolic disorders caused by the high consumption of fructose and saturated fat.

Assessment of the microbiological origin of blowing defects in Grana Padano Protected Designation of Origin cheese.

Recognized worldwide for its history, flavor, and high nutritional quality, Grana Padano (GP) is one of the most traditional Italian raw-milk, hard-cooked, long-ripened cheese. Throughout GP manufacturing, some well-known and undesired bacterial species, such as clostridia, can proliferate and lead to spoilage defects that mischaracterize the final product; however, little is known about the development of late-blowing defects in hard cheese samples without clostridia. Therefore, in this study we aimed to use metataxonomic analysis to identify bacterial taxa associated with the development of late-blowing defect in GP samples. Furthermore, the presence of several heterofermentative lactobacilli species in defective zones were verified by primer-specific PCR assay. Considering α- and ß-diversity analyses, no statistically significant differences were detected between cheese samples with and without blowing defect. Following taxonomic assignment, Lactobacillus and Streptococcus were the dominant genera, whereas clostridia-related taxa were not detected in any of the 20 analyzed samples. Using EdgeR, the genera Propionibacterium and Acinetobacter were found to be prevalently more abundant in samples categorized as having "big regular holes." In samples with "small regular holes," multiplex PCR amplification revealed differences in terms of Lactobacillus population composition, mainly obligate homofermentative lactobacilli, between defective and non-defective zones of the same cheese wheel. This study demonstrated that GP samples with blowing defects not caused by clostridial development share similar biodiversity indices with GP collected from control zones, but an imbalance of obligate homofermentative lactobacilli was noticed between samples, which requires further analysis to better comprehend the exact mechanism involved in this process.

Starmerella bacillaris Released in Vineyards at Different Concentrations Influences Wine Glycerol Content Depending on the Vinification Protocols.

Starmerella bacillaris is a non-Saccharomyces yeast proposed for must fermentation together with Saccharomyces cerevisiae because of its high glycerol and moderate volatile acidity production. Furthermore, it was demonstrated that the same S. bacillaris strains that possess interesting technological properties exhibited antifungal activity against Botrytis cinerea, suggesting the release of this yeast in the vineyard. To obtain a positive effect during the following winemaking process, the maintenance of suitable concentrations of S. bacillaris is essential. Therefore, to obtain information on the survival of S. bacillaris, a small-scale field trial was performed. One week before the harvest, two different concentrations of S. bacillaris (106 and 107 cells/mL) were sprayed on Pinot grigio bunches, and the strain concentration was monitored by means of qPCR during the subsequent fermentation process. In addition, the combined effect of different winemaking techniques was evaluated, i.e., the vinification of juice, juice with marc and cryomaceration treatment. Results demonstrated that, under the tested conditions, S. bacillaris released in the vineyard remained viable for one week on grape bunches and increased glycerol content during the subsequent fermentation process. Different vinification protocols influenced cell concentrations. In particular, the cryomaceration treatment, due to the use of low temperature, supported S. bacillaris growth due to its cryotolerant aptitude. The collected data open new perspectives on the control of alcoholic fermentation, involving both vineyard and cellar management.

Chemoprevention of DMH-Induced Early Colon Carcinogenesis in Male BALB/c Mice by Administration of Lactobacillus Paracasei DTA81.

We evaluated the effects of the probiotic candidate Lactobacillus paracasei DTA81 (DTA81) on liver oxidative stress, colonic cytokine profile, and gut microbiota in mice with induced early colon carcinogenesis (CRC) by 1,2-dimethylhydrazine (DMH). Animals were divided into four different groups (n = 6) and received the following treatments via orogastric gavage for 8 weeks: Group skim milk (GSM): 300 mg/freeze-dried skim milk/day; Group L. paracasei DTA81 (DTA81): 3 × 109 colony-forming units (CFU)/day; Group Lactobacillus rhamnosus GG (LGG): 3 × 109 CFU/day; Group non-intervention (GNI): 0.1 mL/water/day. A single DMH dose (20 mg/kg body weight) was injected intraperitoneally (i.p), weekly, in all animals (seven applications in total). At the end of the experimental period, DTA81 intake reduced hepatic levels of carbonyl protein and malondialdehyde (MDA). Moreover, low levels of the pro-inflammatory cytokines Interleukin-6 (IL-6) and IL-17, as well as a reduced expression level of the proliferating cell nuclear antigen (PCNA) were observed in colonic homogenates. Lastly, animals who received DTA81 showed an intestinal enrichment of the genus Ruminiclostridium and increased concentrations of caecal acetic acid and total short-chain fatty acids. In conclusion, this study indicates that the administration of the probiotic candidate DTA81 can have beneficial effects on the initial stages of CRC development.

Teaching-learning: a mutual exchange between high school and graduate students in the field of microbiology.

The teaching-learning process becomes more attractive when practical classes are used as part of methodological tools. Aiming (i) to stimulate the interest of high school students in the microbiological world and (ii) to provide didactic experience for microbiology graduate students, practical classes were undertaken. These classes were carried out during the years 2016 and 2017, using the infrastructure held at Universidade Federal de Viçosa. Briefly, laboratory classes were prepared and taught by microbiology graduate students and an evaluation questionnaire was answered by the participants after the classes. Approximately 95% of the high school students, from both years, evaluated the experience outside of the school routine as very good and good. A total of 90.09% (2016) and 100% (2017) of graduate students evaluated this experience as very good and good. The relationship between high school and graduate students also increased the curiosity of the former regarding the university environment. Accordingly, the information retention regarding the 'microbiology world' was verified after one year and the students were able to remember important terms related to the microbiology class. In addition, this work allowed graduate students and high school students to build a closer relationship and created an excellent teaching-learning strategy for both.

Microbial Diversity and Nutritional Properties of Persian "Yellow Curd" (Kashk Zard), a Promising Functional Fermented Food.

"Yellow curd" (YC) is one of the most popular homemade Persian fermented foods and is consumed by many people. Notwithstanding, no studies are available to date on its nutritional and microbiological composition. In this study, we examined YC samples obtained from different local markets of Sistan and Baluchestan province, Iran. The results of the chemical analyses revealed a homogenous content of protein (13.71% ± 1.07), lipids (4.09% ± 0.73), and carbohydrates (61% ± 2.13) among the samples. By comparing the average mineral content of YC with yogurt, many relevant differences were detected. Apart from the calcium content, which was similar on average to that of YC, all other minerals tested are present in higher amounts in YC than in yogurt. The analysis of the main sugars present (i.e., lactose, galactose and glucose) highlighted relevant differences among samples, indicating that different YC samples contain natural strains with different capabilities to metabolize sugars. The concentration of galactose in YC samples should be taken into consideration by galactose intolerant people. From the microbiological perspective, the metagenomics analysis revealed that lactic acid bacteria, and particularly the genera Lactobacillus, Pediococcus, and Streptococcus, were dominant in YC. The information provided shows that YC is an interesting base for the preparation of novel functional foods with a good content of beneficial bacteria.

Synbiotic VSL#3 and yacon-based product modulate the intestinal microbiota and prevent the development of pre-neoplastic lesions in a colorectal carcinogenesis model.

Colorectal cancer is a public health problem, with dysbiosis being one of the risk factors due to its role in intestinal inflammation. Probiotics and synbiotics have been used in order to restore the microbiota balance and to prevent colorectal carcinogenesis. We aimed to investigate the effects of the probiotic VSL#3® alone or in combination with a yacon-based prebiotic concentrate on the microbiota modulation and its influence on colorectal carcinogenesis in an animal model. C57BL/6J mice were divided into three groups: control (control diet), probiotic (control diet + VSL#3®), and synbiotic (yacon diet + VSL#3®). The diets were provided for 13 weeks and, from the third one, all animals were subjected to induction of colorectal cancer precursor lesions. Stool samples were collected to evaluate organic acids, feces pH, ß-glucuronidase activity, and microbiota composition. The colon was used to count pre-neoplastic lesions and to determine the cytokines. The microbiota composition was influenced by the use of probiotic and synbiotic. Modifications were also observed in the abundance of bacterial genera with respect to the control group, which confirms the interference of carcinogenesis in the microbiota. Pre-neoplastic lesions were reduced by the use of the synbiotic, but not with the probiotic. The protection provided by the synbiotic can be attributed to the modulation of the intestinal inflammatory response, to the inhibition of a pro-carcinogenic enzyme, and to the production of organic acids. The modulation of the composition and activity of the microbiota contributed to beneficial changes in the intestinal microenvironment, which led to a reduction in carcinogenesis. KEY POINTS: • Synbiotic reduces the incidence of colorectal cancer precursor lesions. • Synbiotic modulates the composition and activity of intestinal microbiota. • Synbiotic increases the abundance of butyrate-producing bacteria.

Genomic and phenotypic assessments of safety and probiotic properties of Streptococcus macedonicus strains of dairy origin.

In this work, we studied the genomes and characterized some probiotic features of four S. macedonicus strains isolated from dairy environments in Italy that already had indicated some technological potential. The genomes of these strains were sequenced and used for genomic in silico studies. All strains were also evaluated for hemolytic activity, susceptibility to most commonly used antibiotics and probiotic potential, such as resistance to simulated gastrointestinal conditions, bile salts hydrolytic activity and adhesion ability to HT-29 human colorectal adenocarcinoma cells. Results revealed that one strain, namely S. macedonicus 211MA, was found to possess probiotic properties, such as resistance to simulated gastrointestinal conditions as well as adherence capability to human epithelial cells. In silico analyses revealed that S. macedonicus 211MA displayed the least number of single copy genes, genomic islands regions and gene content classified as virulence factors when compared to other S. macedonicus and S. gallolyticus strains. Moreover, the maximum gene content associated with bacterial stress response category and the presence of the opuCABCD operon, not detected in the other strains, were correlated with S. macedonicus 211MA capability to resist to low pH and to show higher adhesion to HT-29 human cells. This is the first report on the presence of opuCABCD operon in S. macedonicus and its possible relation with attachment ability and stress response.

Milk microbial composition of Brazilian dairy cows entering the dry period and genomic comparison between Staphylococcus aureus strains susceptible to the bacteriophage vB_SauM-UFV_DC4.

Brazil has the second-largest dairy cattle herd in the world, and bovine mastitis still can cause significant losses for dairy farmers. Despite this fact, little information is available about milk microbial composition of Brazilian dairy cows, as well as the potential use of bacteriophages in the control of S. aureus. Here, we investigated milk bacterial composition of 28 Holstein Fresian cows (109 teats), selected in the dry-off period, using 16S rRNA analysis. Furthermore, a representative S. aureus strain (UFV2030RH1) was obtained at drying-off for isolation of a bacteriophage (vB_SauM-UFV_DC4, UFV_DC4) and bacterial genomic comparison purposes. Our outcomes revealed that Staphylococcus was the third most prevalent genus and positively correlated with subclinical mastitis events. As a major finding, genomic analyses showed the presence of adhesive matrix molecules that recognize microbial surface components (MSCRAMM) in UFV2030RH1 and might indicate great biofilm formation capability. A minimum inhibitory concentration (MIC) assay showed that resistance to ampicillin was the highest among the antibiotic tested in S. aureus 3059 and UFV2030RH1, displaying values four and sixteen times greater than MIC resistance breakpoint, respectively. Together, our results suggest that Staphylococcus is highly prevalent in dairy cows at drying-off and the use of the phage UFV_DC4 as a biocontrol agent must be investigated in future studies.

Safety and Stability of Two Potentially Probiotic Lactobacillus Strains After In Vitro Gastrointestinal Transit.

According to FAO and WHO, probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Most probiotic bacteria used today belong to the genera Lactobacillus and Bifidobacterium and are of animal or human origin. The fundamental characteristic routinely evaluated in potential probiotics strains is their limited viability loss during gastrointestinal transit (GIT), but to date, no studies reported whether probiotics, besides viability, still also maintain their beneficial properties intact. To study this aspect, we considered two strains, Lactobacillus rhamnosus DTA 79 and L. paracasei DTA 83, previously characterised for the presence of some probiotic properties, isolated from faeces of 7- to 21-day-old babies. Here, we examined some additional properties, namely antibiotic resistance, resistance to lysozyme, presence of haemolytic activity and inhibition of pathogen biofilm formation. We then tested the effect of in vitro GIT on all these features and our results show evidence that this procedure had in some cases limited and in others no significant effects on them. Additionally, we examined the gastrointestinal resistance of the strains after skim milk fermentation and successive storage of the product for 20 and 40 days at refrigeration temperature, to see whether prolonged storage could weaken cell resistance to GIT. Our results demonstrate that a protracted refrigeration period before in vitro GIT did not affect or influenced very weakly this essential probiotic property.

Draft genome sequence data of Lactobacillus paracasei strain DTA83 isolated from infant stools.

Here the draft genome sequence of Lactobacillus paracasei strain DTA83, isolated from stools of healthy infants in Rio de Janeiro (Brazil), is reported. The 2.8-Mb genome possesses 2825 protein-coding sequences distributed on 330 SEED subsystems. This strain belongs to a set of potentially probiotic Lactobacillus spp. strains used to study genetic factors related to antibiotic resistance after stress conditions, such as simulated gastrointestinal conditions. The complete genome data have been deposited in GenBank under the accession number QRBH00000000, https://www.ncbi.nlm.nih.gov/nuccore/QRBH00000000.

A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496.

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.

Comparative Transcriptomic Analysis of Streptococcus thermophilus TH1436 and TH1477 Showing Different Capability in the Use of Galactose.

Streptococcus thermophilus is a species widely used in the dairy industry for its capability to rapidly ferment lactose and lower the pH. The capability to use galactose produced from lactose hydrolysis is strain dependent and most of commercial S. thermophilus strains are galactose-negative (Gal-), although galactose-positive (Gal+) would be more technologically advantageous because this feature could provide additional metabolic products and prevent galactose accumulation in foods. In this study, a next generation sequencing transcriptome approach was used to compare for the first time a Gal+ and a Gal- strain to characterize their whole metabolism and shed light on their different properties, metabolic performance and gene regulation. Transcriptome analysis revealed that all genes of the gal operon were expressed very differently in Gal+ and in the Gal- strains. The expression of several genes involved in mixed acid fermentation, PTS sugars transporter and stress response were found enhanced in Gal+. Conversely, genes related to amino acids, proteins metabolism and CRISPR associated proteins were under-expressed. In addition, the strains showed a diverse series of predicted genes controlled by the transcriptional factor catabolite control protein A (CcpA). Overall, transcriptomic analysis suggests that the Gal+ strain underwent a metabolic remodeling to cope with the changed environmental conditions.