Subspecies Lactis Research Articles

Probiotics for managing patients after bowel preparation for colonoscopy: an interventional, double-arm, open, randomized, multi-center, and national study (COLONSTUDY).

Bowel preparation (BP) for colonoscopy induces significant changes in gut microbiota, causing dysbiosis that, in turn, elicits intestinal symptoms. Consequently, probiotics may counterbalance the disturbed microbiota after BP. So, probiotics may restore microbiota homeostasis. The current study evaluated the efficacy and safety of Abincol®, an oral nutraceutical containing a probiotic mixture with Lactobacillus plantarum LP01 (1 billion living cells), Lactobacillus lactis subspecies cremoris LLC02 (800 millions living cells), and Lactobacillus delbrueckii LDD01 (200 millions living cells), Patients were randomized in two groups (2:1). Group A took one stick/daily for four weeks after colonoscopy. Group B was considered as control. Patients were evaluated at baseline (T0) and after one (T1), two (T2), and four (T3) weeks. The severity of symptoms was measured by patients using a Visual Analog Scale. Abincol® significantly diminished the presence and the severity of intestinal symptoms at T2 and even more at T3. All patients well tolerated the probiotic mixture. The present study suggests that Abincol® may be considered an effective and safe therapeutic option in managing patients undergoing BP. The course should last one month.

Effects of Bifidobacterium animalis subsp. lactis BB-12 and yogurt on mice during oral antibiotic administration

Probiotics have the potential to prevent disruptions to normal gastrointestinal function caused by oral antibiotic use. In this study, we examined the capacity of Bifidobacterium animalis subspecies lactis BB-12 (BB-12) and yogurt, separately and combined, to mitigate the effects of the antibiotic amoxicillin-clavulanate (AMC) on the gut microbiota and metabolomes of C57BL/6 J mice. Male and female mice were administered either BB-12, yogurt, BB-12 in yogurt, or saline for 10 days concurrent with the inclusion of AMC in the drinking water. Male mice exposed to AMC exhibited significant reductions (p<0.05) in body weight over the course of the study compared to sham (no AMC) controls whereas no such effects were observed for female mice. AMC administration resulted in rapid alterations to the intestinal microbiota in both sexes irrespective of BB-12 or yogurt treatment, including significant (p<0.05) losses in bacterial cell numbers and changes in microbial alpha-diversity and beta-diversity in the feces and cecal contents. The effects of AMC on the gut microbiota were observed within one day of administration and the bacterial contents continued to change over time, showing a succession marked by rapid reductions in Muribaculaceae and Lachnospiraceae and temporal increases in proportions of Acholeplasmataceae (day 1) and Streptococcaceae and Leuconostocaceae (day 5). By day 10 of AMC intake, high proportions of Gammaproteobacteria assigned as Erwiniaceae or Enterobacteriaceae (average of 63 %), were contained in the stools and were similarly enriched in the cecum. The cecal contents of mice given AMC harbored significantly reduced concentrations of (branched) short-chain fatty acids (SCFA), aspartate, and other compounds, whereas numerous metabolites, including formate, lactate, and several amino acids and amino acid derivatives were significantly enriched. Despite the extensive impact of AMC, starting at day 7 of the study, the body weights of male mice given yogurt or BB-12 (in saline) with AMC were similar to the healthy controls. BB-12 (in saline) and yogurt intake was associated with increased Streptococcaceae and both yogurt and BB-12 resulted in lower proportions of Erwiniaceae in the fecal and cecal contents. The cecal contents of mice fed BB-12 in yogurt contained levels of formate, glycine, and glutamine that were equivalent to the sham controls. These findings highlight the potential of BB-12 and yogurt to mitigate antibiotic-induced gut dysbiosis.

Isolation and identification of Bifidobacterium species from human breast milk and infant feces in Indonesia

Abstract. Dosan R, Mudana SO, Julyanto CMP, Purnama ET, Sugata M, Jo J, Tan TJ. 2024. Isolation and identification of Bifidobacterium species from human breast milk and infant feces in Indonesia. Biodiversitas 25: 337-343. There has been a growing interest in identifying emerging probiotic strains because of their benefits for human health. Many bifidobacteria originated from humans have been reported to possess probiotics properties. They are commonly found in the intestine of breast-fed infants. Hence, this study aimed to isolate and identify bifidobacteria from human breast milk and infant fecal samples in Indonesia and evaluate their probiotic properties. Twenty colonies were isolated from two independent fecal samples and two independent breast milk samples. Ten isolates (BR1-M1, BR1-B1, BR2-5, BR2-6, BR2-12, BS2-PB3, BS2-PB5, BS2-PS1, BS2-PS2, BS2-MB1) showed a compatible phenotypic character with Bifidobacteria based on the Bergey’s Manual, including Gram-positive, irregular rods, no catalase activity, non-spore-forming, and non-motile. Subsequently, four isolates with similar carbohydrate fermentation patterns as Bifidobacterium spp. were selected for further molecular identification based on 16S rRNA gene sequencing analysis. The results showed that BR2-5 and BR2-6 were found to be closely related to Bifidobacterium animalis subspecies lactis with 100 and 98.39% similarity, respectively. Meanwhile, BS2-PS1 and BS2-PB3 were found to be closely related to Bifidobacterium breve with 100 and 98.26% similarity, respectively. Further investigation revealed that BR2-5 and BS2-PB3 were resistant to low pH (?4) and could tolerate the exposure of bile salts (1%). Both isolates survived under different oxidative stress conditions (aerobic and microaerophilic). In conclusion, BR2-5 and BS2-PB3 exhibited promising characteristics as probiotic candidates, though further investigations are required to substantiate these current findings.

Effects of Lactic Acid Bacteria Fermentation on the Physicochemical Properties of Rice Flour and Rice Starch and on the Anti-Staling of Rice Bread.

In this study, Lactococcus lactis lactis subspecies 1.2472, Streptococcus thermophilus 1.2718, and thermostable Lactobacillus rhamnosus HCUL 1.1901-1912 were used to ferment rice flour for preparing rice bread. The characteristics of fermented rice bread were studied to elucidate the mechanism by which fermentation improves the anti-staling ability of rice bread. The amylose content of rice flour increased after fermentation. The peak viscosity, attenuation value, final viscosity, recovery value, and gelatinization temperature decreased. Amylopectin was partially hydrolyzed, and the amylose content decreased. The crystallinity of starch decreased, and the minimum crystallinity of Lactococcus lactis subsp. lactis fermented rice starch (LRS) was 11.64%. The thermal characteristics of fermented rice starch, including To, Tp, Tc, and ΔH, were lower than RS (rice starch), and the △H of LRS was the lowest. Meanwhile, LRS exhibited the best anti-staling ability, and with a staling degree of 43.22%. The T22 of the LRF rice flour dough was lower, and its moisture fluidity was the weakest, indicating that moisture was more closely combined with other components. The texture characteristics of fermented rice bread were improved; among these, LRF was the best: the hardness change value was 1.421 times, the elasticity decrease was 2.35%, and the chewability change was 47.07%. There, it provides a theoretical basis for improving the shelf life of bread.

Bifidobacterium animalis subspecies lactis HN019 can reduce the sequelae of experimental periodontitis in rats modulating intestinal parameters, expression of lipogenic genes, and levels of hepatic steatosis.

To determine whether Bifidobacterium animalis subspecies lactis HN019 (B. lactis HN019) can reduce the sequelae of experimental periodontitis (EP) in rats modulating systemic parameters. This study evaluated the effects of probiotic therapy (PROB) in the prevention of local and systemic damage resulting from EP. Forty-eight rats were allocated into four groups: C (control), PROB, EP, and EP-PROB. PROB (1 × 1010 CFU/mL) administration lasted 8 weeks and PE was induced on the 7th week by placing ligature on the animals' lower first molars. All animals were euthanized in the 9th week of the experiment. Biomolecular analyses, RT-PCR, and histomorphometric analyses were performed. The data obtained were analyzed statistically (ANOVA, Tukey, p < .05). The EP group had higher dyslipidemia when compared to the C group, as well as higher levels of insulin resistance, proteinuria levels, percentages of systolic blood pressure, percentage of fatty hepatocytes in the liver, and expression of adipokines was up-regulated (LEPR, NAMPT, and FABP4). All these parameters (except insulin resistance, systolic blood pressure, LEPR and FABP4 gene expression) were reduced in the EP-PROB group when compared to the EP group. The EP group had lower villus height and crypt depth, as well as a greater reduction in Bacteroidetes and a greater increase in Firmicutes when compared to the EP-PROB group. Greater alveolar bone loss was observed in the EP group when compared to the EP-PROB group. Bifidobacterium lactis HN019 can reduce the sequelae of EP in rats modulating intestinal parameters, attenuating expression of lipogenic genes and hepatic steatosis.

Iranian traditional kefir beverage: isolation and identification of beneficial microorganisms and evaluation of antimicrobial activity against food-borne pathogens

Purpose Kefir is a traditional fermented dairy beverage that has numerous health benefits due to the presence of bacteria and yeasts in an exopolysaccharide matrix. This study aims to isolate and identify beneficial microorganisms and evaluate the antimicrobial activity of kefir beverage against two important food-borne pathogens including Salmonella Typhimurium and Listeria monocytogenes. Design/methodology/approach Microorganisms were identified by the polymerase chain reaction with specific primers, and antimicrobial activity was evaluated by the disk diffusion method. Findings The following microorganisms were identified as natural inhabitants of the kefir grains: Leuconostoc lactis, Lactococcus lactis subspecies lactis, Streptococcus cremoris, Enterococcus faecalis, Enterococcus faecium, Lactobacillus helveticus, Leuconostoc mesenteroides, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium langum, Saccharomyces cerevisiae and Pichia fermentas. Also, the results obtained from the disk diffusion method showed the inhibitory effect of kefir milk on Salmonella Typhimurium and Listeria monocytogenes with an inhibitory average diameter of 8.3 ± 4–9.1 ± 2.8 and 3.4 ± 3–6.6 ± 3 mm, respectively. Originality/value The results of this study showed that Iranian traditional kefir beverage contained different species of lactic acid bacteria and yeasts and has antimicrobial activity against two important food-borne pathogens, Salmonella Typhimurium and Listeria monocytogenes, which the highest inhibitory effect was observed against Salmonella Typhimurium.

Subspecies-level genome comparison of Lactobacillus delbrueckii

Lactobacillus delbrueckii comprises six subspecies, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. jakobsenii, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. sunkii, and L. delbrueckii subsp. indicus. We investigated the evolution of the six subspecies of L. delbrueckii using comparative genomics. While the defining feature of the species was the gene number increment driven by mobile elements and gene fragmentation, the repertoire of subspecies-specific gene gains and losses differed among the six subspecies. The horizontal gene transfer analyses indicated that frequent gene transfers between different subspecies had occurred when the six subspecies first diverged from the common ancestor, but recent gene exchange was confined to a subspecies implying independent evolution of the six subspecies. The subspecies bulgaricus is a homogeneous group that diverged from the other subspecies a long time ago and underwent convergent evolution. The subspecies lactis, jakobsenii, delbrueckii, and sunkii were more closely related to each other than to other subspecies. The four subspecies commonly show increasing genetic variability with increasing genome size. However, the four subspecies were distinguished by specific gene contents. The subspecies indicus forms a branch distant from the other subspecies and shows an independent evolutionary trend. These results could explain the differences in the habitat and nutritional requirements of the subspecies of L. delbrueckii.

Effects of Bifidobacterium animalis subspecies lactis supplementation on gastrointestinal symptoms: systematic review with meta-analysis.

The effects of probiotics on gastrointestinal (GI) symptoms have been increasingly investigated, particularly that of Bifidobacterium animalis. Clinical trials so far have shown differing evidence regarding these effects in healthy adults. To synthesize the published evidence on the effects of B. animalis subspecies lactis on GI symptoms (GIS) in healthy adults. A search of the Medline, Embase, Lilacs, Scopus, Web of Science, ProQuest, and Google Scholar databases was conducted for reports on randomized controlled trials published up to October 2021. Population characteristics and data on colonic transit time (CTT), stool consistency, defecation frequency, abdominal pain, bloating, flatulence, volunteer compliance, and adverse events were extracted. A random-effects model was used to estimate the effect of probiotic treatment on these variables. In total, 1551 studies were identified, of which 14 were included in the qualitative synthesis and 13 in the meta-analysis. Overall, probiotic supplementation increased defecation frequency (standardized mean difference [SMD], 0.26; 95%CI, 0.13-0.39). Subgroup analysis revealed a decrease in CTT (SMD, -0.34; 95%CI, -0.62 to -0.07) in short-term treatment (≤14 d) and an improvement in stool consistency (SMD, 0.76; 95%CI, 0.44-1.08) in individuals without GIS. No improvement in abdominal pain and bloating was found. B. animalis subspecies lactis supplementation may increase defecation frequency and, in short-term treatment, may reduce CTT in healthy adults and improve stool consistency in individuals without GIS. More high-quality randomized controlled trials are needed to develop a clinical protocol for the use of this strain to improve these symptoms. PROSPERO registration no. CRD42020154060.

Changes in the Gut Metabolic Profile of Gestational Diabetes Mellitus Rats Following Probiotic Supplementation.

The roles of gut microbiota and metabolomics in women with gestational diabetes mellitus (GDM) are not well understood. This study investigated the gut metabolomic profiling of GDM rats and GDM rats treated with probiotic supplements. Associations between gut metabolites and microbiota were also studied in GDM rats. Liquid chromatography–mass spectrometry was used to detect gut metabolites in GDM rats and GDM rats treated with probiotic supplements of 0.5 g (low-dose group) or 1 g (high-dose group) for 15 days. Each gram of probiotic supplement contained 5 × 107 colony-forming units (CFU) of Lactobacillus rhamnosus LGG and 1 × 108 CFU of Bifidobacterium animalis subspecies lactis Bb12. The association between gut metabolites and microbiota in GDM rats was investigated using Spearman’s correlation. Finally, 10 rats in the normal pregnant group, eight rats in the GDM model group, eight GDM rats in the low-dose probiotics group, and nine GDM rats in the high-dose probiotics group were further studied. Serum parameters and pancreatic and colon histology were significantly changed in GDM rats, and these were restored using probiotic supplements. In total, 999 gut metabolites were detected in the feces, and GDM rats were distinguished from normal rats. The levels of 44 metabolites were increased in GDM rats, and they were alleviated using probiotic supplements. Changes in metabolites in GDM rats were associated with amino acids and bile acids metabolism signaling pathways. Furthermore, changes in metabolites after probiotic supplementation were associated with porphyrin and chlorophyll metabolism pathways. We found that the Allobaculum genus displayed strong positive correlations, whereas the Bryobacter and Gemmatimonas genera displayed strong negative correlations with metabolisms of amino acids and bile acids in GDM rats. The Lactobacillus and Bifidobacterium genera were positively correlated with gut metabolites. Overall, our results showed that metabolism signaling pathways of amino acids and bile acids are associated with the development of GDM. Probiotic supplements alleviate the pathology of GDM through the metabolism pathways of amino acids, bile acids, porphyrin, and chlorophyll.

Parasite-Probiotic Interactions in the Gut: Bacillus sp. and Enterococcus faecium Regulate Type-2 Inflammatory Responses and Modify the Gut Microbiota of Pigs During Helminth Infection.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Controlled Bio-Fermentation by Lactobacillus and Lactococcus Probiotics for Improving Quality and Safety of Fessiekh (Fermented Grey Mullet)

ABSTRACT In the current study, fessiekh was produced using various probiotics (Lacticaseibacillus casei, Lactobacillus helveticus, and Lactococcus lactis subspecies cremoris) to explore their effect on the quality attributes of fessiekh. Application of bio-fermentation for fessiekh production using probiotics improved the sensory quality attributes and increased the lactic acid bacteria population, with a significant decrease in yellowness (b*) value and anaerobic bacterial count compared to control. The addition of L. casei and L. helveticus during the fermentation of mullet led to a significant decrease in moisture, ash, salt contents, pH, thiobarbituric acid reactive substances, redness (a*) values, S. aureus and mold counts, while protein, fat content, and lightness (L*) value increased compared to L. cremoris and control groups. On the contrary, the redness (a*) value of fessiekh was enhanced more by the application of L. cremoris than Lactobacilli. It was obvious that each probiotic used in mullet fermentation has a special unique effect on fessiekh properties; therefore, the use of probiotics can be a promising procedure to control the fermentation process during fessiekh production and improve the safety and quality of the product.

Oral toxicity evaluation of probiotic strains isolated from Finger millet [Eleusine coracana (L.) Gaertn.] in Wistar rat models (in vivo)

This study evaluates the oral toxicity of five probiotic strains recently isolated from fermented flour of finger-millet (Eleusine coracana) varieties of Sri Lanka. Probiotic strains; Lactobacillus plantarum MF405176, Lactobacillus fermentum MF033346, Lactococcus lactis subspecies lactis MF480428, Enterococcus faecium MF480431and Pediococcus acidilactici MF480434 were evaluated for acute and sub-chronic oral toxicity in Wistars. Three individual doses (108 CFU/g, 1010 CFU/g and 1012 CFU/g) of each probiotic strain at single oral dose of 5000 mg/kg bw were orally administered to rats and observations were done till 14th day. Since no animals demonstrated signs of toxicity as a result of the administrated probiotics strains, repeated dose sub-chronic oral toxicity study was conducted by oral administration of three doses (108 CFU/g, 1010 CFU/g, 1012 CFU/g) of each probiotic strain at 1000 mg/kg bw/day for consecutive 90 days. Administration of probiotic strains to rats did not caused mortality in any of the tested doses. No changes in animal behavior, feed or water intake and negative effects on body weight observed. Probiotic feeding did not cause changes in analyzed biochemical and hematological parameters attributed to toxicity. Bacteremia, bacterial translocation and histopathological changes in rat organs were not observed. No significant difference in liver enzymes observed in treatment groups compared to control. In conclusion, all tested probiotic strains are nonpathogenic therefore could be considered as safe for human consumption.

An adaptable and non-invasive method for tracking Bifidobacterium animalis subspecies lactis 420 in the mouse gut

Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract.

The Industry Originated Probiotic Bacteria Role in COVID-19

Intensive, uncontrolled, long term usage of industry originated Bifidobacterium animalis subspecies lactis by humans lead the “Probiotic contamination” of human gut, when unusual, not common for healthy individuals bacterial colonies have being created and dominate for a long time, changing the function of Human Microbiome. Several strains of Bifidobacterium animalis subspecies lactis (produced by few leading world biotechnological manufacturers) colonize human mucosa surfaces, lead the SARS-CoV-2 increased ability to penetrate Air path and gastrointestinal mucosa surfaces, change an innate immunity of individuals, infect human cells and ensure specific complications, as cytokine storm, atypical pneumonia, clotting. SARS-CoV-2 features (as interaction with ACE2 protein, others possible cell-entry points, binding to human cell surface and endocytosis) are enhanced by above bacterial metabolites. Further on, Bifidobacterium animalis subspecies lactis or metabolically similar one, provide deregulated release of pro and anti-inflammatory cytokines, lead a severe course of COVID-19.

Lactulose ingestion causes an increase in the abundance of gut-resident bifidobacteria in Japanese women: a randomised, double-blind, placebo-controlled crossover trial.

The genus Bifidobacterium comprises various bacterial species, and the complement of species within the human intestinal tract differs from individual to individual. The balance of these bifidobacterial species remains poorly understood, although it is known that the abundance of bifidobacteria increases following the ingestion of prebiotics. We previously conducted a randomised, placebo-controlled, double-blind, crossover study of 2 g/day lactulose ingestion for 2 weeks in 60 Japanese women. To study the effect of lactulose ingestion on each bifidobacterial species, here, we measured the abundance of each of the principal bifidobacterial species. After lactulose ingestion, the log cell counts of the Bifidobacterium adolescentis group (8.97±0.08 vs 9.39±0.08, P=0.0019), Bifidobacterium catenulatum group (9.45±0.10 vs 9.65±0.10, P=0.0032) and Bifidobacterium longum group (9.01±0.07 vs 9.29±0.07, P=0.0012) were significantly higher than in the placebo ingestion control group. However, the log cell counts were similar for Bifidobacterium breve (8.12±0.12 vs 8.33±0.12, P=0.20), Bifidobacterium bifidum (9.08±0.12 vs 9.42±0.14, P=0.095) and Bifidobacterium animalis subspecies lactis (8.65±0.53 vs 8.46±0.46, P=0.77). Cluster analysis of the log cell count data at the bifidobacterial species level revealed three distinct clusters, but the combinations and ratios of the constituent bifidobacteria were not affected by lactulose ingestion. Furthermore, principal coordinate analysis of the intestinal microbiota in the lactulose and placebo ingestion groups using Illumina MiSeq showed no significant differences in the intestinal microbiota as a whole. These results suggest that 2 g/day lactulose ingestion for 2 weeks significantly increases the abundance of intestinal bifidobacteria, but does not affect the intestinal microbiota as a whole.

LACTOCOCCUS LACTIS STRAINS FROM INTESTINAL ORGAN OF BLACK TIPS SHARK CARCHARHINUS LIMBATUS PRODUCING NISIN-LIKE BACTERIOCIN ACTIVE AGAINST SHRIMP AND FISH PATHOGENS (VIBRIO PARAHAEMOLYTICUS AND VIBRIO ALGINOLYTICUS)

Shark can be a potential source for host-associated probiotics destined to be used in aquaculture. In this study, intestinal tissues of Black tip shark (Carcharhinus limbatus) was used as the source sample for inhibition screening based on spot-on-lawn and agar well diffusion methods. Out of the 80 isolates, only four showed antagonistic activities against selected indicator strains. All of the 4 isolates FA1, FA2, FA3 and FA4 were found to be Gram positive coccus, non-spore former, oxidase and catalase negatives, as well as lactose fermenters. The isolate FA1, FA2, FA3 and FA4 were found to demonstrate broad range of inhibitory spectrum on gram-positive indicator bacterium (Staphylococcus aureus and Bacillus cereus) and the gram-negative bacterium (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Vibrio alginolyticus and Vibrio parahaemolyticus); with the highest inhibition zone at 20.0±0.1 mm, recorded on V. parahaemolyticus. The genotype of the isolates was characterized using 16S rRNA sequencing. Each sequence with a given GenBank (NCBI) accession number (MN975529 for FA1; MN982712 for FA2; MN982711 for FA3; and MN982710 for FA4) showed at least 99% similarity with Lactococcus lactis subspecies. The crude cell free supernatant (CFS) of the isolates potentially contained a putative bacteriocin displaying nisin-like properties. The pH and catalase treatments showed that neither organic acid nor hydrogen peroxide (H2O2) was the inhibitory component. An almost 95% inactivation of the CFS’s antimicrobial activity was observed following treatment with trypsin suggested a presence of proteinaceous agent. Growth and inhibition studies on all isolated strains demonstrated that the inhibition properties were growth associated, with maximum inhibition achieved at ~20.0 ± 0.1 mm (or ~168 ± 17 AU/ml) at 17 hours by FA2 strain against V. parahaemolyticus. A study on the mode of inhibition demonstrated a bactericidal killing against V. parahaemolyticus. The isolation of Lactococcus lactis strain from shark is rather unique since it was commonly reported to be isolated from human and animals. These strains show bacteriocinogenic properties, broad spectrum inhibition, and more importantly, they are able to antagonise some pathogens implicated in fish or shrimp diseases. Therefore, these strains have potential for use as probiotics in aquaculture.

Precise Populations’ Description in Dairy Ecosystems Using Digital Droplet PCR: The Case of L. lactis Group in Starters

Lactococcus lactis group (composed of the lactis and cremoris subspecies, recently reassigned as two distinct species) plays a major role in dairy fermentations. Usually present in starter cultures, the two species enable efficient acidification and improve the organoleptic qualities of the final product. Biovar diacetylactis strains produce diacetyl and acetoin, aromas from the citrate metabolization. As these populations have distinct genomic and phenotypic characteristics, the proportions of each other will affect the final product. Today, there is no quantitative test able to distinguish between the two species and the biovar in dairy ecosystems. In this study, we developed a specific, reliable, and accurate strategy to quantify these populations using, species-, and diacetylactis-specific fluorescent probes in digital droplet PCR assays (ddPCR). Species were distinguished based on three single nucleotide polymorphisms in the glutamate decarboxylase gadB gene, and the citD gene involved in citrate metabolism was used to target the biovar. Used in duplex or singleplex, these probes made it possible to measure the proportion of each population. At 59°C, the probes showed target specificity and responded negatively to the non-target species usually found in dairy environments. Depending on the probe, limit of detection values in milk matrix ranged from 3.6 × 103 to 1.8 × 104 copies/ml. The test was applied to quantify sub-populations in the L. lactis group during milk fermentation with a commercial starter. The effect of temperature and pH on the balance of the different populations was pointed out. At the initial state, lactis and cremoris species represent, respectively, 75% and 28% of the total L. lactis group and biovar diacetylactis strains represent 21% of the lactis species strains. These ratios varied as a function of temperature (22°C or 35°C) and acidity (pH 4.5 or 4.3) with cremoris species promoted at 22°C and pH4.5 compared to at 35°C. The biovar diacetylactis strains were less sensitive to acid stress at 35°C. This methodology proved to be useful for quantifying lactis and cremoris species and biovar diacetylactis, and could complete 16S metagenomics studies for the deeply description of L. lactis group in complex ecosystems.

Effect of Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies lactis BB-12 combination on the duration of diarrhea and length of hospital stay in children with acute diarrhea in Turkey

Background Acute diarrhea continues to be a leading cause of morbidity and mortality worldwide. The main therapy for all individuals with dehydration caused by diarrhea is oral rehydration. Probiotics have been proposed as a complementary therapy in the treatment of acute diarrhea. We aim to evaluate the effect of a combination of Lactobacillus rhamnosus GG (LGG) with Bifidobacterium animalis subspecies lactis BB-12 (BB-12) on the duration of diarrhea and length of hospital stay in children with acute diarrhea.Methods A multicenter, randomized (240 children, 2:1 randomized for probiotic vs. control), single blind, hospital based clinical trial was performed in children (6 to 60 months) with acute watery diarrhea lasting more than 24 but less than 72 hours, requiring hospitalization. We enrolled children with clinical signs of mild to moderate dehydration. The children received conventional therapy with or without the combination of LGG and BB-12 (1 × 109 colony forming units for each) for 5 days. The primary endpoint was the duration of diarrhea (in hours), defined as the first normal stool according to the Bristol stool score (score &amp;lt; 5). Secondary outcome measures were duration of hospitalization (days) and percentage of children without diarrhea at 72 hours of intervention.Results In total, data from 218 of 240 children could be evaluated (150 in the probiotic group and 68 in the control group). The duration of diarrhea was significantly reduced in the LGG and BB-12 group compared to the control group (74.5 ± 40.8 hours vs. 98.4 ± 22.9 hours, P&amp;lt;0.001). The percentage of diarrhea-free children was significantly larger in the LGG and BB-12 group at 72 hours compared to the control (60% vs. 33.8%, P&amp;lt;0.001). Mean length of hospital stay was similar for both groups (5.03 ± 2.3 days vs. 5.25 ± 1.3 days, P&amp;gt;0.05).Conclusion This is the first clinical trial to test the combination of LGG and BB-12, and show its effects on diarrhea duration in children with acute infectious diarrhea. The duration of diarrhea was reduced by approximately 24 hours in the hospitalized children. Further randomized controlled clinical trials including outpatient cases with acute infectious diarrhea in addition to hospitalized cases should be conducted to assess the potential effects of the combination in more detail.

Probiotics Reduce Mortality and Morbidity in Preterm, Low-Birth-Weight Infants: A Systematic Review and Network Meta-analysis of Randomized Trials

We aimed to compare the effectiveness of single- vs multiple-strain probiotics in a network meta-analysis of randomized trials. We searched MEDLINE, Embase, Science Citation Index Expanded, CINAHL, Scopus, Cochrane CENTRAL, BIOSIS Previews, and Google Scholar through January 1, 2019, for studies of single-strain and multistrain probiotic formulations on the outcomes of preterm, low-birth-weight neonates. We used a frequentist approach for network meta-analysis and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the certainty of evidence. Primary outcomes included all-cause mortality, severe necrotizing enterocolitis (NEC) (Bell stage II or more), and culture-proven sepsis. We analyzed data from 63 trials involving 15,712 preterm infants. Compared with placebo, a combination of 1 or more Lactobacillus species (spp) and 1 or more Bifidobacterium spp was the only intervention with moderate- or high-quality evidence of reduced all-cause mortality (odds ratio [OR], 0.56; 95% confidence interval [CI], 0.39-0.80). Among interventions with moderate- or high-quality evidence for efficacy compared with placebo, combinations of 1 or more Lactobacillus spp and 1 or more Bifidobacterium spp, Bifidobacterium animalis subspecies lactis, Lactobacillus reuteri, or Lactobacillus rhamnosus significantly reduced severe NEC (OR, 0.35 [95% CI, 0.20-0.59]; OR, 0.31 [95% CI, 0.13-0.74]; OR, 0.55 [95% CI, 0.34-0.91]; and OR, 0.44 [95% CI, 0.21-0.90], respectively). There was moderate- or high-quality evidence that combinations of 1 or more Lactobacillus spp and 1 or more Bifidobacterium spp and Saccharomyces boulardii reduced the number of days to reach full feeding (mean reduction of 3.30 days [95% CI, reduction of 5.91-0.69 days]). There was moderate- or high-quality evidence that, compared with placebo, the single-species product B animalis subsp lactis or L reuteri significantly reduced duration of hospitalization (mean reduction of 13.00 days [95% CI, reduction of 22.71-3.29 days] and mean reduction of 7.89 days [95% CI, reduction of 11.60-4.17 days], respectively). In a systematic review and network meta-analysis of studies to determine the effects of single-strain and multistrain probiotic formulations on outcomes of preterm, low-birth-weight neonates, we found moderate to high evidence for the superiority of combinations of 1 or more Lactobacillus spp and 1 or more Bifidobacterium spp vs single- and other multiple-strain probiotic treatments. The combinations of Bacillus spp and Enterococcus spp, and 1 or more Bifidobacterium spp and Streptococcus salivarius subsp thermophilus, might produce the largest reduction in NEC development. Further trials are needed.

Use of a probiotic mixture containing Bifidobacterium animalis subsp. lactis BB-12 and Enterococcus faecium L3 as prophylaxis to reduce the incidence of acute gastroenteritis and upper respiratory tract infections in children.

For healthy children, attending communities such as nurseries, kindergartens or schools, exposes them to the risk of acute gastroenteritis (AGE) and/or upper respiratory tract infections (URTIs). We therefore evaluated whether the use of a well-documented probiotic formula could act as prophylaxis for AGE and URTIs, reducing the risk of occurrence. In a randomized study, we tested a probiotic mixture containing Bifidobacterium animalis subspecies lactis BB-12 and Enterococcus faecium L3 on 94 healthy children, comparing the incidence and duration of episodes of AGE and the incidence of URTIs to those of a control group of 109 healthy, untreated subjects. In a subgroup consisting of 34 healthy, treated children, we also evaluated salivary IgA levels. The use of the probiotic formula significantly reduced the incidence and duration of episodes of AGE by 82% and 45%, respectively, and the incidence and duration of episodes of URTIs by 84% and 50%. Salivary IgA levels significantly increased three-fold after 90 days of probiotic treatment. The probiotic formula was well tolerated and no side effects occurred. According to our results, use of the probiotic strains BB-12 and L3 statistically reduced the risk of AGE and URTIs in healthy children and increased levels of salivary IgA.