Soft Hydrogel Actuator for Fast Machine-Learning-Assisted Bacteria Detection.

We demonstrate that our bio-electrochemical platform facilitates the reduction of detection time from the 3-day period of the existing tests to 15 min. Machine learning and robotized bioanalytical platforms require the principles such as hydrogel-based actuators for fast and easy analysis of bioactive analytes. Bacteria are fragile and environmentally sensitive microorganisms that require a special environment to support their lifecycles during analytical tests. Here, we develop a bio-electrochemical platform based on the soft hydrogel/eutectic gallium-indium alloy interface for the detection of Streptococcus thermophilus and Bacillus coagulans bacteria in various mediums. The soft hydrogel-based device is capable to support bacteria' viability during detection time. Current-voltage data are used for multilayer perceptron algorithm training. The multilayer perceptron model is capable of detecting bacterial concentrations in the 104 to 108 cfu/mL range of the culture medium or in the dairy products with high accuracy (94%). Such a fast and easy biodetection is extremely important for food and agriculture industries and biomedical and environmental science.

Rapid isolation of exopolysaccharide-producing Streptococcus thermophilus based on molecular marker screening.

BACKGROUND: As a natural food additive, exopolysaccharide (EPS) produced by Streptococcus thermophilus can improve product viscosity and texture. The protein EpsA is a putative pathway-specific transcriptional regulator for EPS biosynthesis in S. thermophilus. RESULTS: According to comparative analysis of EPS biosynthetic gene clusters, a conserved region of epsA (609 bp) was employed to design primer pair epsA-F/R as a molecular marker for the isolation of EPS-producing (EPS+ ) S. thermophilus. Two EPS+ S. thermophiles strains, AR333 and S-3, were band-positive, whereas Lactococcus lactis NZ9000 (non-EPS-producing, EPS- ), Lactobacillus casei LC2W (EPS+ ) and L. plantarum AR113 (EPS+ ) were negative by polymerase chain reaction (PCR) amplicon bands using the epsA probe. This indicated good specificity of the epsA probe to EPS+ S. thermophilus. Moreover, based on PCR screening with the epsA probe, 23 positive strains were isolated and identified as S. thermophilus from our microbial library and natural fermented milk with 141.3-309.2 mg L-1 of EPS production, demonstrating the validity of our molecular marker screening method. CONCLUSION: The designed molecular marker of epsA can rapidly screen EPS+ S. thermophilus, which has potential application in the dairy and other food industries. © 2021 Society of Chemical Industry.

Microbial exopolysaccharide production of Streptococcus thermophilus and its antiquorum sensing activity.

Interest in the production of exopolysaccharides by microorganisms has increased in the recent years. Using low-cost product is the main step of microbial production to reduce cost and compete with chemical production. In this work, EPS production of Streptococcus thermophilus isolates from yogurt (S2), kefir (S3), and S. thermophilus ATCC 19258 (S1) isolate which was used as control strains were investigated by using different fruit pulps. S. thermophilus isolates were identified by morphological and 16S sequence analysis. The amount of EPS obtained was measured spectrophotometrically using glucose as standard with phenol sulfuric acid method. All three isolates produced higher amounts of EPS on M17 medium than Nutrient medium. When the fruit pulp was added to the medium, EPS production increased in all three isolates. When different nitrogen sources were added together with fruit pulp juice, EPS production increased. The highest amount of EPS produced by ATCC 19258 strain (21.570 mg/L) and S3 isolate (29.131 mg/L) is the medium where mixed fruit pulp juice and nitrogen source is tryptophan. It has been shown that EPS production is increased by adding fruit pulps to the prepared media. It is thought that apricot pulp can be a good alternative in EPS production especially in the evaluation of wastes. Also, antiquorum sensing activity of the highest amount EPS was determined by using Chromobacterium violaceum CV026 strain and found effective on violacein pigment inhibition and C6-AHL production of biosensor strain.

Microbiological, Physicochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt.

Nut-based milks and yogurts are gaining popularity, but may not offer the same benefits as dairy yogurts to consumers. Cashew nuts often cause severe allergic reactions, and cashew nut allergens are stable to several types of processing. To compare its characteristics to dairy yogurt and characterize the effects of fermentation on the Ana o 1-3 cashew nut allergens, a commercial yogurt made from cashew nuts (Cashewgurt) was evaluated for microbiological, physiochemical, and immunological properties. Average counts for lactobacilli and Streptococcus thermophilus were greater than 10 million colony forming units per milliliter, indicating the capacity to provide a health benefit. Cashewgurt pH and viscosity values were comparable to cow milk yogurts, and it was off white in color. SDS-PAGE analysis indicated a clear reduction in Ana o 1 and 2, and immuno-assay with polyclonal anti-cashew IgG antibody and cashew-allergic IgE indicated an overall reduction in allergen content. In contrast, SDS-PAGE, mass spectrometry, immunoblot, and ELISA all revealed that Ana o 3 was relatively unaffected by the fermentation process. In conclusion, Ana o 1 and Ana o 2 are sensitive to degradation, while Ana o 3 survives lactic acid bacterial fermentation during yogurt production. The analysis presented here indicates that cashew nut yogurt is not suitable for those with cashew nut allergy.

Large-scale genome-wide analysis links lactic acid bacteria from food with the gut microbiome.

Lactic acid bacteria (LAB) are fundamental in the production of fermented foods and several strains are regarded as probiotics. Large quantities of live LAB are consumed within fermented foods, but it is not yet known to what extent the LAB we ingest become members of the gut microbiome. By analysis of 9445 metagenomes from human samples, we demonstrate that the prevalence and abundance of LAB species in stool samples is generally low and linked to age, lifestyle, and geography, with Streptococcus thermophilus and Lactococcus lactis being most prevalent. Moreover, we identify genome-based differences between food and gut microbes by considering 666 metagenome-assembled genomes (MAGs) newly reconstructed from fermented food microbiomes along with 154,723 human MAGs and 193,078 reference genomes. Our large-scale genome-wide analysis demonstrates that closely related LAB strains occur in both food and gut environments and provides unprecedented evidence that fermented foods can be indeed regarded as a possible source of LAB for the gut microbiome.

Microbiological characterization of Gioddu, an Italian fermented milk.

Gioddu, also known as "Miciuratu", "Mezzoraddu" or "Latte ischidu" (literally meaning acidulous milk), is the sole variety of traditional Italian fermented milk. The aim of the present study was to elucidate the microbiota and the mycobiota occurring in artisan Gioddu sampled from three Sardinian producers by combining the results of viable counting on selective culture media and high-throughput sequencing. Physico-chemical parameters were also measured. The overall low pH values (3.80-4.22) recorded in the analyzed Gioddu samples attested the strong acidifying activity carried out by lactic acid bacteria during fermentation. Viable counts revealed the presence of presumptive lactococci, presumptive lactobacilli and non-Saccharomyces yeasts. A complex (kefir-like) microbiota of bacteria and yeasts was unveiled through sequencing. In more detail, Lactobacillus delbrueckii was found to dominate in Gioddu together with Streptococcus thermophilus, thus suggesting the establishment of a yogurt-like protocooperation. Unexpectedly, in all the three analyzed batches from two out of the three producers Lactobacillus kefiri was also detected, thus representing an absolute novelty, which suggests the presence of bioactive compounds (e.g. exopolysaccharides) similar to those characterizing milk kefir beverage. Mycobiota population, studied for the very first time in Gioddu, revealed a more complex composition, with Kluyveromyces marxianus, Galactomyces candidum and Geotrichum galactomyces constituting the core species. Further research is needed to disclose the eventual occurence in Gioddu of probiotic cultures and bioactive compounds (e.g. exopolysaccharides, angiotensin-converting enzyme inhibitory peptides and antimicrobial compounds) with potential health-benefits for the consumers.

Safety and technological application of autochthonous Streptococcus thermophilus cultures in the buffalo Mozzarella cheese.

Thermophilic and mesophilic lactic acid bacteria (LAB), such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, and Lactococcus lactis, play a crucial role in the technological and sensory quality of Mozzarella cheese. In this study, the safety (genes encoding virulence factors and antibiotic resistance) and acidifying activity of autochthonous S. thermophilus cultures were evaluated in order to choose the most suitable strain for industrial application. The safe and good acidifying culture was tested in two buffalo Mozzarella cheese batches: Mozzarella cheeses produced with autochthonous culture (SJRP107) and commercial culture (STM5). The cultivable LAB was evaluated by culture-dependent method (plate counting) and the quantification of S. thermophilus cultures (commercial and autochthonous) were evaluated by culture-independent method RealT-qPCR (real-time quantitative polymerase chain reaction). The texture, physicochemical and proteolytic properties of the Mozzarella cheeses were similar for both batches. The nonstarter LAB count was higher during manufacture than in the storage, and the RealT-qPCR indicated the presence of S. thermophilus culture until the end of storage. S. thermophilus SJRP107 presented high potential for safety application in the production of Mozzarella cheese. Furthermore, considering the culture characteristics and their relationship with product quality, further studies could be helpful to determine their effect on the sensory characteristics of the cheese.

First Insight into the Probiotic Properties of Ten Streptococcus thermophilus Strains Based on In Vitro Conditions.

The aim of this study was to evaluate probiotic properties of ten Streptococcus thermophilus strains (st1 to st10) isolated from pickles in China. These strains all had ß-galactosidase activity, which laid foundation for studying their probiotic properties. In this study, the bile salt hydrolase activity, lysozyme resistance, tolerance to simulated gastric juice, bile salt tolerance, and bacterial adhesion capacity to the Caco-2 cells of these selected strains were detected in vitro conditions. The results indicated that the bile salt hydrolase activities of st2, st6, and st9 were higher than that for other strains. St10 showed the greatest lysozyme resistance (> 80% survival), followed by st9, st8, st7, st5, and st6. As for the tolerance to simulated gastric juice, st5 possessed the highest survival rate (35%), followed by st6 (30%). St6 was the best performer in both bile salt tolerance and bacterial adhesion capacity to the Caco-2 cells. The results of fluorescence microscope and electron microscope further confirmed previous studies and more intuitively demonstrated the st6 strain's tolerance to harsh environments. Overall, these strains were expected to possess beneficial properties and have the potentiality to be probiotics.

PacBio sequencing reveals bacterial community diversity in cheeses collected from different regions.

Cheese is a fermented dairy product that is popular for its unique flavor and nutritional value. Recent studies have shown that microorganisms in cheese play an important role in the fermentation process and determine the quality of the cheese. We collected 12 cheese samples from different regions and studied the composition of their bacterial communities using PacBio small-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA). Our data revealed 144 bacterial genera (including Lactobacillus, Streptococcus, Lactococcus, and Staphylococcus) and 217 bacterial species (including Lactococcus lactis, Streptococcus thermophilus, Staphylococcus equorum, and Streptococcus uberis). We investigated the flavor quality of the cheese samples using an electronic nose system and we found differences in flavor-quality indices among samples from different regions. We found a clustering tendency based on flavor quality using principal component analysis. We found correlations between lactic acid bacteria and the flavor quality of the cheese samples. Biodegradation and metabolism of xenobiotics, and lipid-metabolism-related pathways, were predicted to contribute to differences in cheese flavor using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). This preliminary study explored the bacterial communities in cheeses collected from different regions and their potential genome functions from the perspective of flavor quality.

A survey of the relationship between functional genes and acetaldehyde production characteristics in Streptococcus thermophilus by multilocus sequence typing.

Streptococcus thermophilus is an important bacterium used in the production of fermented dairy products. Yogurt with good flavor is preferred by consumers; thus, variation in flavor-formation characteristics among isolates is attracting attention. Here, acetaldehyde production characteristics of 30 isolates were evaluated in parallel with genotyping and multilocus sequence typing of key functional genes involved in acetaldehyde production. The results showed that isolates could be divided into 3 phenotypically distinct groups: high-acetaldehyde-yielding isolates (>10 mg/L), medium-acetaldehyde-yielding isolates (5-10 mg/L) and low-acetaldehyde-yielding (<5 mg/L) based on evaluation of acetaldehyde production during yogurt storage. These groups, distinguishable by phenotypic characteristics, were clustered in corresponding groups based on functional gene multilocus sequence typing analysis. Combining functional gene sequence analysis of 30 Strep. thermophilus isolates with phenotypic evaluation of their flavor-related characteristics (specifically acetaldehyde production) demonstrated that groups of isolates established using genotype data analysis corresponded with groups identified based on their phenotypic traits. Interestingly, the 30 isolates of Strep. thermophilus showed significant phylogenetic clustering in acetaldehyde content by functional gene and acetaldehyde content analysis. A corresponding relationship exists between functional gene phylogenetic clustering and acetaldehyde content variation.

Colonization of the human gut by bovine bacteria present in Parmesan cheese.

The abilities of certain microorganisms to be transferred across the food production chain, persist in the final product and, potentially, colonize the human gut are poorly understood. Here, we provide strain-level evidence supporting that dairy cattle-associated bacteria can be transferred to the human gut via consumption of Parmesan cheese. We characterize the microbial communities in samples taken from five different locations across the Parmesan cheese production chain, confirming that the final product contains microorganisms derived from cattle gut, milk, and the nearby environment. In addition, we carry out a human pilot study showing that Bifidobacterium mongoliense strains from cheese can transiently colonize the human gut, a process that can be enhanced by cow milk consumption.

Characterization and transfer of antimicrobial resistance in lactic acid bacteria from fermented dairy products in China.

INTRODUCTION: Lactic acid bacteria (LAB) are commonly found in foods and are also natural intestinal inhabitants in humans and most animals. However, information regarding antimicrobial resistance and the transfer of resistance genes of LAB from fermented dairy products in China is limited. METHODOLOGY: In this study, LAB isolates (n = 82) of Lactobacillus (n = 43) and Streptococcus thermophilus (n = 39) were isolated from 51 commercial fermented food samples in China. All isolates were subjected to pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility, detecting resistance genes, as well as investigating the transferability of resistance genes. RESULTS: The 43 Lactobacillus isolates yielded 24 PFGE patterns and the 34 isolates of S. thermophilus generated 32 different PFGE patterns. Among the 43 Lactobacillus strains, the most commonly observed resistance was that to streptomycin (83.7%) and gentamycin (83.7%). Among the 39 S. thermophilus strains, the most frequently observed resistance was that to streptomycin (92.3%), gentamycin (87.2%), ciprofloxacin (79.5%), and chloramphenicol (71.8%), whereas the lowest level of resistance was that against erythromycin (7.7%). Antimicrobial resistance genes for erythromycin (emrB), gentamycin (aac(6')-aph(2")), streptomycin (ant(6)), sulfamethoxazole (sulI and sulII), tetracycline (tetM and tetS) were detected in the 18 resistance LAB strains. Conjugation experiments showed that tetM from L. delbrueckii subsp. bulgaricus R6 and tetS from L. plantarum R41 were successfully transferred to L. monocytogenes by filter mating. CONCLUSIONS: LAB strains could potentially act as reservoirs of resistance genes and play an active role in the transfer of resistance to humans via the food chain.

Antibiotic Resistance of Lactobacillus spp. and Streptococcus thermophilus Isolated from Chinese Fermented Milk Products.

The aim of the present study was to investigate the phenotypic and genotypic antimicrobial resistance and the transferability of resistance markers in 87 lactic acid bacterial strains recovered from fermented milk products obtained from different areas of China. The isolates were identified as 21 Lactobacillus bulgaricus, 8 Lactobacillus casei, 6 Lactobacillus rhamnosus, 3 Lactobacillus paracasei, 2 Lactobacillus acidophilus, and 47 Streptococcus thermophilus strains. High levels of intrinsic resistance were revealed among the tested species. The following resistance genes were detected in strains isolated from fermented milk products: tet(M) in two L. bulgaricus and two S. thermophilus isolates, strA and strB in nine and seven S. thermophilus isolates, respectively; sul1 in six L. bulgaricus and seven S. thermophilus isolates, sul2 in one S. thermophilus isolate, aac(6')-aph(2″) in two L. bulgaricus isolates, and aph(3″)-II and aph(3″)-III in one S. thermophilus and two L. bulgaricus isolates, respectively. Transfer of the monitored antibiotic resistance genes was not observed in the filter mating assays of this study. To our knowledge, the strA, strB, sul1, sul2, and aph(3″)-II genes in S. thermophilus, and the sul1 and aac(6')-aph(2″) genes in L. bulgaricus were identified for the first time. These results indicate the potential risks posed by lactic acid bacteria (LAB) in fermented milk products in expanding the antibiotic resistance gene reservoir and transferring antibiotic resistance genes among bacteria. Further investigations are required to identify the potential sources of contamination and the dissemination routes of antibiotic resistance genes among LAB in fermented milk products.

Gut microbiota of preterm infants supplemented with probiotics: sub-study of the ProPrems trial.

BACKGROUND: The ProPrems trial, a multi-center, double-blind, placebo-controlled randomized trial, previously reported a 54% reduction in necrotizing enterocolitis (NEC) of Bell stage 2 or more from 4.4 to 2.0% in 1099 infants born before 32 completed weeks' gestation and weighing < 1500 g, receiving probiotic supplementation (with Bifidobacterium longum subsp. infantis BB-02, Streptococcus thermophilus TH-4 and Bifidobacterium animalis subsp. lactis BB-12). This sub-study investigated the effect of probiotic supplementation on the gut microbiota in a cohort of very preterm infants in ProPrems. RESULTS: Bifidobacterium was found in higher abundance in infants who received the probiotics (AOR 17.22; 95% CI, 3.49-84.99, p < 0.001) as compared to the placebo group, and Enterococcus was reduced in infants receiving the probiotic during the supplementation period (AOR 0.27; 95% CI, 0.09-0.82, p = 0.02). CONCLUSION: Probiotic supplementation with BB-02, TH-4 and BB-12 from soon after birth increased the abundance of Bifidobacterium in the gut microbiota of very preterm infants. Increased abundance of Bifidobacterium soon after birth may be associated with reducing the risk of NEC in very preterm infants.

Differences in Carbohydrates Utilization and Antibiotic Resistance Between Streptococcus macedonicus and Streptococcus thermophilus Strains Isolated from Dairy Products in Italy.

Streptococcus thermophilus and S. macedonicus are the only two species of the genus related to food productions so far known. In the present study, eight S. thermophilus and seven S. macedonicus strains isolated from dairy environments in Italy were compared in order to evidence possible species-specific technological characteristics. Their capability to use lactose, galactose, fructose, and glucose, sugars commonly present in foods and two carbohydrates considered as prebiotics, xylose and inulin, along with the respective growth kinetics were studied. Results showed a luxuriant growth on lactose and different behaviors on galactose, glucose, and fructose. No growth on inulin and xylose was recorded, which is a positive feature for strains intended to be used as starter cultures. Growth parameters, namely, λ, µmax, and Nmax, were estimated by using the Gompertz model. Antibiotic resistance to 14 drugs revealed an overall similar behavior between the two species with only a marked difference regarding gentamycin. Antimicrobial activity was also tested against six deleterious bacterial strains, but none of the strains evidenced inhibitory capabilities. The results presented here could be helpful to compare technological potentialities of the two species and to choose strains of the most suitable species for selected microbiological food transformations.

Short communication: Genomic and phenotypic analyses of exopolysaccharides produced by Streptococcus thermophilus KLDS SM.

Streptococcus thermophilus plays important roles in the dairy industry. Streptococcus thermophilus KLDS SM could produce a high amount of exopolysaccharides (EPS). To understand the possible link between the genotype and the phenotype regarding EPS, the complete genome of S. thermophilus KLDS SM was sequenced and investigated in silico for genes related to carbohydrate fermentation, nucleotide sugars synthesis, and EPS gene cluster. We found that S. thermophilus KLDS SM is able to ferment sucrose, mannose, glucose, galactose, and lactose from the genomic research, which was confirmed by API 50 CH (bioMérieux, Marcy l'Etoile, France). The genetic analysis of nucleotide sugars and EPS cluster revealed that the EPS produced by this strain are composed of galactose and glucose, in accordance with the biochemical result. Furthermore, differences in the molecular mass of EPS from S. thermophilus KLDS SM cultivated under different carbon sources were correlated with the transcription levels of the genes encoding chain length determination protein and glycosyltransferase. Our findings provide a better understanding of the link between the genetic elements and the chemical conformation of EPS and a theoretical basis for producing tailor-made EPS through genetic and metabolic engineering approaches.

Folates biosynthesis by Streptococcus thermophilus during growth in milk.

The ability of folate-producer strains of Streptococcus thermophilus to accumulate folates and the expression of two target genes (folK and folP), involved in the folate biosynthesis, were studied during milk fermentation. An over-expression of folK took place only in the early phase of growth, whereas folP was mainly expressed in the mid log-phase of growth and declined thereafter. The accumulation of total folates, which was quantified by a microbiological assay, was strain-dependent. Two major forms of folates, i.e. tetrahydrofolate (THF) and 5-methyl-tetrahydrofolate (5-Met-THF), were identified and quantified by HPLC. With respect to the level accumulated by a weak folate producer (St 383), used as calibrator in the expression experiments and as control in folate quantification in milk, the strains St 563 and St 399 produced 5-Met-THF in amounts significantly higher than THF. The possibility of using selected folate-producer S. thermophilus strains as functional cultures for a bio-fortification of dairy products is discussed.

Lactobacillus plantarum and Streptococcus thermophilus as starter cultures for a donkey milk fermented beverage.

Donkey milk is recently gaining attention due to its nutraceutical properties. Its low casein content does not allow caseification, so the production of a fermented milk would represent an alternative way to increase donkey milk shelf life. The aim of this study was to investigate the possibility of employing selected Streptococcus thermophilus and Lactobacillus plantarum isolates for the production of a novel donkey milk fermented beverage. Lysozyme resistance and the ability to acidify donkey milk were chosen as main selection parameters. Different fermented beverages (C1-C9) were produced, each with a specific combination of isolates, and stored at refrigerated conditions for 35days. The pH values and viability of the isolates were weekly assessed. In addition, sensory analysis was performed. Both S. thermophilus and L.plantarum showed a high degree of resistance to lysozyme with a Minimum Bactericidal Concentration>6.4mg/mL for 100% of S. thermophilus and 96% of L. plantarum. S. thermophilus and L. plantarum showed the ability to acidify donkey milk in 24h at 37°C, with an average ΔpH value of 2.91±0.16 and 1.78±0.66, respectively. Four L. plantarum and two S. thermophilus were chosen for the production of fermented milks. Those containing the association S. thermophilus/L. plantarum (C1-C4) reached a pH lower than 4.5 after 18h of fermentation and showed microbial loads higher than 7.00logcfu/mL until the end of the storage period. Moreover, comparing the microbial loads of samples containing both species and those containing S. thermophilus alone (C5), we highlighted the ability of L. plantarum to stimulate S. thermophilus replication. This boosted replication of S. thermophilus allowed to reach an appropriate pH in a time frame fitting the production schedule. This was not observed for samples containing a single species (C5-C9). Thus, L. plantarum strains seem to be good candidates in the production of a novel type of fermented milk, not only for their probiotic potential, but also for the enhancing effect on S. thermophilus growth.

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer.

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by whole-genome sequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3' end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appear particularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcus faecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase.IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restriction-modification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating that diffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present.

Evaluation of qPCR and plate counting for quantifying thermophilic starters in cheese.

The respective inputs of plate counting and qPCR for the quantification of starters in cheese were evaluated using hard-cooked cheeses made with various starter combinations. Five starter strains were quantified at their different growth phases, from 0.5 h to day 214 of manufacture: one strain of Streptococcus thermophilus (ST) and two strains each of Lactobacillus delbrueckii (LD) and Lactobacillus helveticus (LH). Numbers of colony-forming units (CFU) were obtained by plate counting (PC) and qPCR (GNCFU). The qPCR standard curves require a special attention since GNCFU depends on the degree of culturability of the standard culture. Discrepancies were evidenced from the vat milk to the end of ripening. During cheese making, GNCFU were lower than PC at the inoculation for all ST and LD samples and 83% of the LH samples, and during both the exponential and stationary phases for many of the ST and LD samples. During ripening which corresponds to the decline phase, GNCFU were higher than PC for 90% of the ST, 78% of the LD and 69% of the LH samples. Hypotheses are discussed to explain those discrepancies. The data provided by GNCFU and PC complement each other, providing a better description of starter growth in cheese.