Probiotic-Induced Modulation of Microbiota Composition and Antibiotic Resistance Genes Load, an In Vitro Assessment.

The imbalance of the gut microbiota (GM) is known as dysbiosis and is associated with disorders such as obesity. The increasing prevalence of microorganisms harboring antibiotic resistance genes (ARG) in the GM has been reported as a potential risk for spreading multi-drug-resistant pathogens. The objective of this work was the evaluation, in a fecal culture model, of different probiotics for their ability to modulate GM composition and ARG levels on two population groups, extremely obese (OB) and normal-weight (NW) subjects. Clear differences in the basal microbiota composition were observed between NW and OB donors. The microbial profile assessed by metataxonomics revealed the broader impact of probiotics on the OB microbiota composition. Also, supplementation with probiotics promoted significant reductions in the absolute levels of tetM and tetO genes. Regarding the blaTEM gene, a minor but significant decrease in both donor groups was detected after probiotic addition. A negative association between the abundance of Bifidobacteriaceae and the tetM gene was observed. Our results show the ability of some of the tested strains to modulate GM. Moreover, the results suggest the potential application of probiotics for reducing the levels of ARG, which constitutes an interesting target for the future development of probiotics.

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin.

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.

Inhibition of platelet-surface-bound proteins during coagulation under flow I: TFPI.

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Tissue factor pathway inhibitor (TFPI) is one such inhibitor, well known for its inhibitory action on the active enzyme complex comprising tissue factor (TF) and activated clotting factor VII. This complex forms when TF embedded in the blood vessel wall is exposed by injury and initiates coagulation. A different role for TFPI, independent of TF:VIIa, has recently been discovered whereby TFPI binds a partially cleaved form of clotting factor V (FV-h) and impedes thrombin generation on activated platelet surfaces. We hypothesized that this TF-independent inhibitory mechanism on platelet surfaces would be a more effective platform for TFPI than the TF-dependent one. We examined the effects of this mechanism on thrombin generation by including the relevant biochemical reactions into our previously validated mathematical model. Additionally, we included the ability of TFPI to bind directly to and inhibit platelet-bound FXa. The new model was sensitive to TFPI levels and, under some conditions, TFPI could completely shut down thrombin generation. This sensitivity was due entirely to the surface-mediated inhibitory reactions. The addition of the new TFPI reactions increased the threshold level of TF needed to elicit a strong thrombin response under flow, but the concentration of thrombin achieved, if there was a response, was unchanged. Interestingly, we found that direct binding of TFPI to platelet-bound FXa had a greater anticoagulant effect than did TFPI binding to FV-h alone, but that the greatest effects occurred if both reactions were at play. The model includes activated platelets' release of FV species, and we explored the impact of varying the FV/FV-h composition of the releasate. We found that reducing the zymogen FV fraction of this pool, and thus increasing the fraction that is FV-h, led to acceleration of thrombin generation.

Transcriptome Modifications in the Porcine Intramuscular Adipocytes during Differentiation and Exogenous Stimulation with TNF-α and Serotonin.

Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the 'PPAR signaling' and 'insulin resistance' pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of 'GPCR ligand-binding', 'cell chemotaxis', 'blood coagulation and complement', 'metabolism of lipid and lipoproteins', 'regulation of lipid metabolism by PPARA', and 'lipid digestion, mobilization and transport' pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.

The citrus flavanone naringenin attenuates zymosan-induced mouse joint inflammation: induction of Nrf2 expression in recruited CD45+ hematopoietic cells.

BACKGROUND: Naringenin is a biologically active analgesic, anti-inflammatory, and antioxidant flavonoid. Naringenin targets in inflammation-induced articular pain remain poorly explored. METHODS: The present study investigated the cellular and molecular mechanisms involved in the analgesic/anti-inflammatory effects of naringenin in zymosan-induced arthritis. Mice were pre-treated orally with naringenin (16.7-150 mg/kg), followed by intra-articular injection of zymosan. Articular mechanical hyperalgesia and oedema, leucocyte recruitment to synovial cavity, histopathology, expression/production of pro- and anti-inflammatory mediators and NFκB activation, inflammasome component expression, and oxidative stress were evaluated. RESULTS: Naringenin inhibited articular pain and oedema in a dose-dependent manner. The dose of 50 mg/kg inhibited leucocyte recruitment, histopathological alterations, NFκB activation, and NFκB-dependent pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-33), and preproET-1 mRNA expression, but increased anti-inflammatory IL-10. Naringenin also inhibited inflammasome upregulation (reduced Nlrp3, ASC, caspase-1, and pro-IL-1ß mRNA expression) and oxidative stress (reduced gp91phox mRNA expression and superoxide anion production, increased GSH levels, induced Nrf2 protein in CD45+ hematopoietic recruited cells, and induced Nrf2 and HO-1 mRNA expression). CONCLUSIONS: Naringenin presents analgesic and anti-inflammatory effects in zymosan-induced arthritis by targeting its main physiopathological mechanisms. These data highlight this flavonoid as an interesting therapeutic compound to treat joint inflammation, deserving additional pre-clinical and clinical studies.

Clinical Impact of Aprepitant in Patients Receiving High-Dose Chemotherapy prior to Autologous Peripheral Blood Stem Cell Transplantation: A Cost-Effectiveness Analysis.

OBJECTIVE: To evaluate the clinical and cost benefits of the administration of aprepitant for the prophylaxis of chemotherapy-induced nausea and vomiting (CINV) during high-dose chemotherapy (HDCT). METHODS: We retrospectively reviewed the charts of patients who received HDCT at our institution between January 2009 and December 2013. Cost-effectiveness was analyzed using direct medical costs. RESULTS: We identified a total of 38 patients (27 with non-Hodgkin lymphoma and 11 with multiple myeloma). Thirteen patients received aprepitant and granisetron (aprepitant group) for CINV prophylaxis, whereas 25 patients received granisetron only (non-aprepitant group). The incidence of severe nausea (≥grade 3) was significantly lower in the aprepitant group than in the non-aprepitant group (p = 0.039). The total mean cost per patient during hospitalization, excluding the cost of HDCT and transplantation, was USD 10,941.8 in the aprepitant group and USD 14,577.2 in the non-aprepitant group (p = 0.041). This cost benefit reflected reductions in the costs of hospitalization, transfusion, and infection treatment. CONCLUSIONS: Our data indicated that the addition of aprepitant for CINV prophylaxis during HDCT reduced the incidence of severe nausea and might also provide economic benefit in the overall management of HDCT prior to autologous peripheral blood stem cell transplantation.

Probiotics modulate gut microbiota and health status in Japanese cedar pollinosis patients during the pollen season.

BACKGROUND: Japanese cedar pollinosis (JCP) is a challenging public health problem in Japan. Altered gut microbiota is associated with several diseases, including allergic diseases. However, only a few studies have focused on JCP and the underlying mechanisms for probiotic effects remain unclear. In addition, this study is the first observation of the correlation between the gut microbiota and blood lipid in JCP. METHODS: Faecal samples from JCP subjects were collected before and after treatment with (n = 14) and without (n = 11) LGG-TMC0356-fermented milk for 10 weeks. Gut microbiota composition was characterized from faecal DNA using sequencing of 16S rRNA genes. RESULTS: 16S rRNA-based operational taxonomic unit clustering of the microbiota revealed that LGG-TMC0356-fermented milk significantly altered gut microbiota after 10 weeks of milk consumption, and eight dominant genera of microbes were detected. During the JCP season, the Bacteroidetes/Firmicutes ratio, when compared to baseline, was significantly decreased in subjects at end of the study. Bacteroidetes showed positive correlation with LDL- and HDL-cholesterol levels, whereas Firmicutes showed negative correlation with total cholesterol, LDL- and HDL- cholesterol. CONCLUSIONS: The altered gut microbiota through supplementation of fermented milk containing the study probiotics may be a prospective target for protection against JCP, with beneficial effects on blood lipid levels.

Quercetin inhibits gout arthritis in mice: induction of an opioid-dependent regulation of inflammasome.

We investigated the anti-inflammatory and analgesic effects of quercetin in monosodium urate crystals (MSU)-induced gout arthritis, and the sensitivity of quercetin effects to naloxone, an opioid receptor antagonist. Mice were treated with quercetin, and mechanical hyperalgesia was assessed at 1-24 h after MSU injection. In vivo, leukocyte recruitment, cytokine levels, oxidative stress, NFκB activation, and gp91phox and inflammasome components (NLRP3, ASC, Pro-caspase-1, and Pro-IL-1ß) mRNA expression by qPCR were determined in the knee joints at 24 h after MSU injection. Inflammasome activation was determined, in vitro, in lipopolysaccharide-primed macrophages challenged with MSU. Quercetin inhibited MSU-induced mechanical hyperalgesia, leukocyte recruitment, TNFα and IL-1ß production, superoxide anion production, inflammasome activation, decrease of antioxidants levels, NFκB activation, and inflammasome components mRNA expression. Naloxone pre-treatment prevented all the inhibitory effects of quercetin over MSU-induced gout arthritis. These results demonstrate that quercetin exerts analgesic and anti-inflammatory effect in the MSU-induced arthritis in a naloxone-sensitive manner.

A combination of probiotics and whey proteins enhances anti-obesity effects of calcium and dairy products during nutritional energy restriction in aP2-agouti transgenic mice.

Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409, Streptococcus thermophilus TMC1543 and whey proteins were used to prepare fermented milk. For the experiment aP2- agouti transgenic mice were pre-treated with a high-sucrose/high-fat diet for 6 weeks to induce obesity. The obese mice were fed a diet containing 1·2% Ca and either non-fat dried milk (NFDM) or probiotic-fermented milk (PFM) with nutritional energy restriction for 6 weeks. The animals were examined after the treatment for changes in body weight, fat pad weight, fatty acid synthase (FAS) activity, lypolysis, the expression levels of genes related to lipid metabolism, insulin sensitivity in adipocytes and skeletal muscle and the presence of biomarkers for oxidative and inflammatory stress in plasma. It was found that the PFM diet significantly reduced body weight, fat accumulation, and adipocyte FAS activity, and increased adipocyte lipolysis as compared with the effects of the NFDM diet (P<0·05). The adipose tissue gene expression of 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) was significantly suppressed in mice that were fed PFM as compared with those that were fed NFDM (P<0·05). PFM caused a greater up-regulation of skeletal muscle PPARα, PPARδ, uncoupling protein 3 (UCP3) and GLUT4 expression and a significant decrease in the plasma concentration of insulin, malondialdehyde, TNF-α, monocyte chemotactic protein-1 and C-reactive protein as compared with the effects of NFDM (P<0·05). Fermentation of milk with selected probiotics and supplementation of milk with whey proteins may thus enhance anti-obesity effects of Ca and dairy products by the suppression of adipose tissue lipogenesis, activation of fat oxidation in skeletal muscle and reduction of oxidative and inflammatory stress.

Influence of orally administered Lactobacillus GG on respiratory immune response in a murine model of diet-induced obesity.

Mice with diet-induced obesity were fed with Lactobacillus rhamnosus GG (LGG) suspended in saline or saline alone (control mice). Pulmonary mRNA expression of IFN-γ; IFN-α receptor 1; CD247 antigen; killer cell lectin-like receptor subfamily K, member 1; TNF-α; IL-12 receptor ß1 and IL-2 receptor ß, and the proportion of Lactobacillales in feces were significantly greater in the LGG group than in the control mice (P < 0.05 and P < 0.01, respectively). These results suggest that LGG alters the respiratory immunity of obese subjects through having a potent impact on intestinal immunity.

Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor.

BACKGROUND: Fermented milk is considered one of the best sources for efficient consumption of probiotic strains by hosts to promote good health. The purpose of this study was to investigate the effects of orally administering LGG-fermented milk (LGG milk) on intestinal inflammation and injury and to study the mechanisms of LGG milk's action. METHODS: LGG milk and non-LGG-fermented milk (non-LGG milk) were administered through gavage to mice before and during dextran sodium sulfate (DSS)-induced intestinal injury and colitis. Inflammatory/injury score and colon length were assessed. Intestinal epithelial cells were treated with the soluble fraction of LGG milk to detect its effects on the epidermal growth factor receptor (EGFR) and its downstream target, Akt activation, cytokine-induced apoptosis, and hydrogen peroxide (H2O2)-induced disruption of tight junctions. RESULTS: LGG milk treatment significantly reduced DSS-induced colonic inflammation and injury, and colon shortening in mice, compared to that in non-LGG milk-treated and -untreated mice. The soluble fraction of LGG milk, but not non-LGG milk, stimulated the activation of EGFR and Akt in a concentration-dependent manner, suppressed cytokine-induced apoptosis, and attenuated H2O2-induced disruption of tight junction complex in the intestinal epithelial cells. These effects of LGG milk were blocked by the EGFR kinase inhibitor. LGG milk, but not non-LGG milk, contained two soluble proteins, p40 and p75, that have been reported to promote survival and growth of intestinal epithelial cells through the activation of EGFR. Depletion of p40 and p75 from LGG milk abolished the effects of LGG milk on prevention of cytokine-induced apoptosis and H2O2-induced disruption of tight junctions. CONCLUSIONS: These results suggest that LGG milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins.

Effects of heat-inactivated Lactobacillus gasseri TMC0356 on metabolic characteristics and immunity of rats with the metabolic syndrome.

The present study investigated the potential health-promoting effects of heat-inactivated Lactobacillus gasseri TMC0356 (TMC0356) on the metabolic syndrome (MS) and the probable mechanisms underlying these effects using an MS rat model. For the purpose of the study, sixty Sprague-Dawley rats were randomly divided into five groups: a control group fed a conventional diet, an MS model group fed a high-fat and high-salt (HFS) diet and three TMC0356 test groups (low-, medium- and high-dose groups) fed an HFS diet supplemented with TMC0356 at 41.8, 83.5 and 167.0 mg/kg body weight (BW) per d, respectively. Food intake and BW were measured weekly. Fasting blood glucose (FBG), lipid profiles and blood pressure (BP) were measured at 0, 5, 10 and 15 weeks. Organ coefficients, immune cell counts and serum insulin, adiponectin, C-reactive protein (CRP), IL-6, TNF-α, IgG and secretory IgA levels were measured at the 15th week after diet intervention. The HFS diet increased the BW, liver or fat:BW ratio, FBG, homeostatic model assessment of insulin resistance, adiponectin, serum LDL-cholesterol and total cholesterol levels and BP (P< 0.01). Average food and energy intakes in the three TMC0356 groups were significantly lower than those of the MS model group. All the metabolic indices, except BP, were markedly improved (P< 0.05) by oral administration of low and medium doses of TMC0356. The thymus index in the medium-dose group and lymphocyte, CRP, IL-6, TNF-α and IgG levels in all the three TMC0356 groups were significantly increased (P< 0.05 or P< 0.01) compared with those in the MS model group. These results suggest that TMC0356 can improve the metabolic characteristics of MS rats by suppressing appetite. Additionally, the enhancement of inflammatory immune response may be, at least in part, the mechanism underlying the health-promoting effects of TMC0356 on the MS.

Development of a compact wireless Laplacian electrode module for electromyograms and its human interface applications.

In this study, we developed a compact wireless Laplacian electrode module for electromyograms (EMGs). One of the advantages of the Laplacian electrode configuration is that EMGs obtained with it are expected to be sensitive to the firing of the muscle directly beneath the measurement site. The performance of the developed electrode module was investigated in two human interface applications: character-input interface and detection of finger movement during finger Braille typing. In the former application, the electrode module was combined with an EMG-mouse click converter circuit. In the latter, four electrode modules were used for detection of finger movements during finger Braille typing. Investigation on the character-input interface indicated that characters could be input stably by contraction of (a) the masseter, (b) trapezius, (c) anterior tibialis and (d) flexor carpi ulnaris muscles. This wide applicability is desirable when the interface is applied to persons with physical disabilities because the disability differs one to another. The investigation also demonstrated that the electrode module can work properly without any skin preparation. Finger movement detection experiments showed that each finger movement was more clearly detectable when comparing to EMGs recorded with conventional electrodes, suggesting that the Laplacian electrode module is more suitable for detecting the timing of finger movement during typing. This could be because the Laplacian configuration enables us to record EMGs just beneath the electrode. These results demonstrate the advantages of the Laplacian electrode module.

Potent effects of, and mechanisms for, modification of crosstalk between macrophages and adipocytes by lactobacilli.

The murine macrophage-like cell line J774.1 was treated with heat-killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)-6, IL-12, and tumor necrosis factor-α were profiled from the J774.1 cells using enzyme-linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3-L1 (which is a mouse embryonic fibroblast-adipose-like cell line). Growth and differentiation of 3T3-L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator-activated receptor (PPAR)-γ mRNA. The medium conditioned by 3T3-L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS-conditioned medium, lipid accumulation in 3T3-L1 cells was significantly suppressed in a dose-dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR-γ mRNA expression in 3T3-L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3-L1 adipose phenotype significantly stimulated production of IL-6 and IL-12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.

Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells.

Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

The gut microbiota of non-obese Japanese pregnant women with gestational diabetes mellitus.

Recent evidence has shown that gut microbiota dysbiosis is associated with development of gestational diabetes mellitus (GDM). However, the gut microbiota composition of non-obese women with GDM, which accounts for a relatively large percentage of Asian GDM, is unknown. We investigated the characteristics of gut microbiota of Japanese pregnant women with GDM. Fecal samples from Japanese pregnant women with GDM (n=20) and normal glucose tolerance (NGT, n=16) were collected at the time of GDM diagnosis (T1), at 35-37 weeks of gestation (T2), and at 4 weeks postpartum (T3). Gut microbiota composition was characterized from fecal DNA by sequencing of 16S rRNA genes. Serum samples were collected late in the third trimester, and the circulating levels of adiponectin and IL-6 were measured by ELISA. At the genus level, Peptostreptococcaceae Romboutsia was enriched in GDM women at T1 (p=0.008) and T2 (p=0.047). The women with lower serum adiponectin tended to have more Romboutsia. The Shannon index was significantly lower in the GDM women at T3 than in the NGT women (p=0.008), and that of the GDM women decreased significantly from T2 to T3 (p=0.02). No significant difference in bacterial community structure was found in a beta diversity analysis. The non-obese GDM women (body mass index <25.0 kg/m2) showed a lower abundance of Coriobacteriaceae Collinsella at T1 (p=0.03) and higher abundance of Akkermansia at T2 (p=0.04) than the normal control. The non-obese GDM women had the distinctive gut microbiota profiles. Analysis of gut microbiota is potentially useful for risk assessment of GDM in non-obese pregnant women.

Effect of Probiotic Bifidobacterium bifidum TMC3115 Supplementation on Psychosocial Stress Using a Sub-Chronic and Mild Social Defeat Stress in Mice.

With the accumulation of knowledge on the relation between psychological stress and gut microbiota, there is growing interest in controlling stress and/or mood disorders via probiotic supplementation. We aimed to examine the effect of probiotic Bifidobacterium bifidum TMC3115 (TMC3115) supplementation using a sub-chronic and mild social defeat stress murine model in this study. TM3115 supplementation maintained body weight gain and alleviated a polydipsia-like symptom induced by the stress. In the analyses of fecal and cecal bacterial profiles, expansions of Proteobacteria in stressed mice and increases in Actinobacteria and Bifidobacterium in mice supplemented with TMC3115 were observed. There was no marked difference in the diversity of cecal bacteria between the tested mice. Elevated serum levels of inflammatory markers such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 were observed in the stressed mice, while TMC3115 only reduced the IL-6 level. These findings suggest that TMC3115 supplementation confers tolerance to psychosocial stress in the host through modulation of the gut microbiota and alleviation of stress-induced inflammatory responses. Furthermore, it may be expected to exert prevention and treatment of disorders related to peripheral IL-6, including depression.

Dysbiosis of Ocular Surface Microbiota in Patients With Refractive Allergic Conjunctival Diseases.

PURPOSE: We investigated ocular surface microbiota dysbiosis in patients with refractory allergic conjunctival diseases (ACDs; stratified into mild and severe groups) treated with topical tacrolimus. METHODS: Patients (n = 21) with refractory ACDs (including vernal and atopic keratoconjunctivitis) actively treated with topical tacrolimus and 6 healthy controls were evaluated. Based on clinical scores and expression of specific cytokines on the ocular surface, patients with ACDs were divided into mild and severe groups using cluster analysis. The microbial composition of tear specimens collected from patients with mild and severe ACD and control subjects using the Schirmer test paper was determined through next-generation 16S rRNA sequencing analysis. RESULTS: Compared with healthy controls, patients with ACDs exhibited significantly decreased ocular surface microbiota α-diversity. Ocular surface microbiota mainly comprised members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria in all groups. The relative abundance of ocular surface microbiota in patients with ACDs was increased for phylum Firmicutes and decreased for phylum Proteobacteria (compared with control subjects). The genera Blautia (vs. mild ACD group) and Morganella (vs. control group) exhibited significantly increased abundance only in the severe ACD group. CONCLUSIONS: The ocular surface microbiota in patients with severe ACD exhibited decreased diversity and exacerbation of dysbiosis compared with that in patients with mild ACD and control subjects. Patients with mild refractory ACD also exhibited decreased diversity of these microbiota. These alterations in microbiota indicated a change in the ocular surface of patients with refractory ACD (be it because of disease pathogenesis or topical immunomodulatory treatment).

Strain-specific detection by pulsed-field gel electrophoresis of Lactobacillus gasseri TMC0356 in human feces after oral administration of these organisms.

The present study aimed to develop an innovative, strain-specific means of identifying the probiotic Lactobacillus gasseri TMC0356 and to determine whether orally administered TMC0356 could be recovered from the human intestine. High molecular weight genomic DNA was isolated from TMC0356 and 14 reference strains of L. gasseri, including the type strain. The DNA samples were digested with the selected rare-cutting restriction endonucleases SmaI, SacII and ApaI and the resulting fragments separated by pulsed-field gel electrophoresis (PFGE) in a size range between 20 to 290 kb. TMC0356 could be distinguished from the other L. gasseri strains on the basis of the SmaI and SacII macrorestriction patterns. Furthermore, L. gasseri strains isolated from the feces of subjects who had ingested TMC0356 were identical to TMC0356 in the SmaI, SacII and ApaI macrorestriction fragments of digested DNA. These results suggest that PFGE of genomic DNA digested with SmaI, SacII, could be a practical means of identification of TMC0356. Furthermore, these results indicate that ingested TMC0356 can survive in, and colonize, the human intestine.