Staphylococcus hsinchuensis sp. nov., Isolated from Soymilk.

A novel coagulase-negative Staphylococcus strain (H164T) was isolated from soymilk in Taiwan. Comparative sequence analysis of the 16S rRNA gene revealed that the H164T strain is a member of the genus Staphylococcus. We used multilocus sequence analysis (MLSA) and phylogenomic analyses to demonstrate that the novel strain was closely related to Staphylococcus gallinarum, Staphylococcus nepalensis, Staphylococcus cohnii, and Staphylococcus urealyuticus. The average nucleotide identity and digital DNA-DNA hybridization values between H164T and its closest relatives were <95% and <70%, respectively. The H164T strain could also be distinguished from its closest relatives by the fermentation of d-fructose, d-maltose, d-trehalose, and d-mannitol, as well as by the activities of α-glucosidase and alkaline phosphatase. The major cellular fatty acids were C15:0 iso and C15:0 anteiso, and the predominant menaquinones were MK-7 and MK-8, respectively. The major cellular fatty acids and predominant menaquinones were C15:0 iso and C15:0 anteiso and MK-7 and MK-8, respectively. In conclusion, this strain represents a novel species, named Staphylococcus hsinchuensis sp. nov., with the type strain H164T (=BCRC 81404T = NBRC 116174T).

Selection of Fermentation Supernatant from Probiotic Strains Exhibiting Intestinal Epithelial Barrier Protective Ability and Evaluation of Their Effects on Colitis Mouse and Weaned Piglet Models.

The intestinal epithelial barrier can prevent the invasion of pathogenic microorganisms and food antigens to maintain a consistent intestinal homeostasis. However, an imbalance in this barrier can result in various diseases, such as inflammatory bowel disease, malnutrition, and metabolic disease. Thus, the aim of this study was to select probiotic strains with epithelial barrier-enhancing ability in cell-based model and further investigate them for their improving effects on colitis mouse and weaned piglet models. The results showed that selected specific cell-free fermentation supernatants (CFSs) from Ligilactobacillus salivarius P1, Lactobacillus gasseri P12, and Limosilactobacillus reuteri G7 promoted intestinal epithelial cell growth and proliferation, strengthening the intestinal barrier in an intestinal epithelial cell line Caco-2 model. Further, the administration of CFSs of L. salivarius P1, L. gasseri P12, and L. reuteri G7 were found to ameliorate DSS-induced colitis in mice. Additionally, spray-dried powders of CFS from the three strains were examined in a weaned piglet model, only CFS powder of L. reuteri G7 could ameliorate the feed/gain ratio and serum levels of D-lactate and endotoxin. In conclusion, a new potential probiotic strain, L. reuteri G7, was selected and showed ameliorating effects in both colitis mouse and weaned piglet models.

A Heat-Killed Probiotic Mixture Regulates Immune T Cells Balance and IgE Production in House Dust Mite Extraction-Induced Atopic Dermatitis Mice.

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease accompanied with severe itching and skin lesions. Current studies have demonstrated that probiotics can exert an immunomodulatory effect, improve epithelial barrier function, and normalize the composition of gut microbiota. Thus, the aim of this study was to investigate the effect of probiotics on the immune balance of AD in vivo. We first screened two lactic acid bacteria strains, which were Lactococcus lactis subsp. cremoris MP01 and Lactobacillus paracasei subsp. paracasei MP02, from 10 strains isolated from traditional fermented milk with inflammation regulating activities in vitro. In the house dust mite (HDM) extraction-induced AD mouse model, mice were assigned randomly to four groups: control group (PC), HDM-induced AD group (NC), HDM-induced AD mice with administration of a mixture of heat-killed MP01 and MP02 at a low concentration (LD), and high concentration (HD) groups. Compared with the NC group, the probiotic treatments could relieve the AD symptoms. Moreover, the LD group significantly decreased total and HDM-specific IgE concentration. These results indicated that a combination of heat-killed MP01 and MP02 strains modulated the proportion of IL4+CD4+ T cells and IFNγ+CD4+ T cells in the spleen of HDM extraction-induced AD mice. In conclusion, administration of the heat-killed MP01 and MP02 mixtures appeared to relieve the classic AD signs, decrease serum IgE concentration, and rebalance the population of Th1/Th2 cells in HDM extraction-induced AD mice. The immunomodulatory activities of a combination of heat-killed MP01 and MP02 provided a potential new therapeutic strategy against AD.

The Antimicrobial Effects of Bacterial Cellulose Produced by Komagataeibacter intermedius in Promoting Wound Healing in Diabetic Mice.

As a conventional medical dressing, medical gauze does not adequately protect complex and hard-to-heal diabetic wounds and is likely to permit bacterial entry and infections. Therefore, it is necessary to develop novel dressings to promote wound healing in diabetic patients. Komagataeibacter intermedius was used to produce unmodified bacterial cellulose, which is rarely applied directly to diabetic wounds. The produced cellulose was evaluated for wound recovery rate, level of inflammation, epidermal histopathology, and antimicrobial activities in treated wounds. Diabetic mices' wounds treated with bacterial cellulose healed 1.63 times faster than those treated with gauze; the values for the skin indicators in bacterial cellulose treated wounds were more significant than those treated with gauze. Bacterial cellulose was more effective than gauze in promoting tissue proliferation with more complete epidermal layers and the formation of compact collagen in the histological examination. Moreover, wounds treated with bacterial cellulose alone had less water and glucose content than those treated with gauze; this led to an increase of 6.82 times in antimicrobial protection, lower levels of TNF-α and IL-6 (39.6% and 83.2%), and higher levels of IL-10 (2.07 times) than in mice wounds treated with gauze. The results show that bacterial cellulose produced using K. intermedius beneficially affects diabetic wound healing and creates a hygienic microenvironment by preventing inflammation. We suggest that bacterial cellulose can replace medical gauze as a wound dressing for diabetic patients.

Lactobacillus rhamnosus Strain LRH05 Intervention Ameliorated Body Weight Gain and Adipose Inflammation via Modulating the Gut Microbiota in High-Fat Diet-Induced Obese Mice.

SCOPE: This study aims to investigate the underlying mechanism of a specific probiotic strain on suppression of adipogenesis and inflammatory response in white adipose tissue (WAT) of high-fat diet (HFD)-fed mice. METHODS AND RESULTS: Eight strains are screened in vitro for candidates of potential probiotics. Lactobacillus rhamnosus LRH05 (LRH05) and Lactobacillus reuteri LR47 (LR47) are screened out with lower triglyceride expression in vitro. The mice are fed a control diet (CD), HFD, or HFD supplemented with a dose of LRH05 or LR47 at 109 CFU per mouse per day for 10 weeks (n = 8), respectively. The results demonstrate that LRH05, but not LR47, significantly reduce body weight gain and the weight of WAT, as well as improve hepatic steatosis and glucose intolerance. LRH05 regulates the Mogat1, Igf-1, Mcp-1, and F4/80 mRNA expression and decreases macrophage infiltration in WAT. LRH05 shows an increase in butyric and propionic acid-producing bacteria, including Lachnoclostridium, Romboutsia, and Fusobacterium that is coincident with the increased fecal propionic acid and butyric acid levels. CONCLUSION: LRH05 shows a strain-specific effect on ameliorating the pro-inflammatory process by reducing inflammatory macrophage infiltration and the expression of inflammation-related genes in mice. Thus, LRH05 can be considered a potential probiotic strain to prevent obesity.

AB-Kefir Reduced Body Weight and Ameliorated Inflammation in Adipose Tissue of Obese Mice Fed a High-Fat Diet, but Not a High-Sucrose Diet.

Consumption of different types of high-calorie foods leads to the development of various metabolic disorders. However, the effects of multi-strain probiotics on different types of diet-induced obesity and intestinal dysbiosis remain unclear. In this study, mice were fed a control diet, high-fat diet (HFD; 60% kcal fat and 20% kcal carbohydrate), or western diet (WD; 40% kcal fat and 43% kcal carbohydrate) and administered with multi-strain AB-Kefir containing six strains of lactic acid bacteria and a Bifidobacterium strain, at 109 CFU per mouse for 10 weeks. Results demonstrated that AB-Kefir reduced body weight gain, glucose intolerance, and hepatic steatosis with a minor influence on gut microbiota composition in HFD-fed mice, but not in WD-fed mice. In addition, AB-Kefir significantly reduced the weight and size of adipose tissues by regulating the expression of CD36, Igf1, and Pgc1 in HFD-fed mice. Although AB-Kefir did not reduce the volume of white adipose tissue, it markedly regulated CD36, Dgat1 and Mogat1 mRNA expression. Moreover, the abundance of Eubacterium_coprostanoligenes_group and Ruminiclostridium significantly correlated with changes in body weight, liver weight, and fasting glucose in test mice. Overall, this study provides important evidence to understand the interactions between probiotics, gut microbiota, and diet in obesity treatment.

Investigating the Mechanistic Differences of Obesity-Inducing Lactobacillus kefiranofaciens M1 and Anti-obesity Lactobacillus mali APS1 by Microbolomics and Metabolomics.

Many studies have investigated the anti-obesity effects of probiotics in animal models and humans. However, few studies have focused on the mechanisms of obesity-inducing probiotics. In a previous study, we demonstrated that specific bacterial strains isolated from kefir, Lactobacillus kefirnofaciens M1 and Lactobacillus mali APS1, possess obesity and anti-obesity effects, respectively, in high-fat diet (HFD)-induced obese mice. Thus, in the present study, we systematically investigated whether APS1 and M1 affect energy homeostasis and lipid metabolism in HFD-induced obese mice and how this might be achieved. We observed that the M1/APS1 intervention influenced fat accumulation by regulating adipogenesis and inflammation-related marker expression both in vitro and in a HFD induced C57BL/6J mice model. We also observed putative links between key taxa and possible metabolic processes of the gut microbiota. Notably, families Christensenellaceae and S24_7 were negatively correlated with body weight gain through increase in the essential esterized carnitine for energy expenditure. These results suggest the importance of specific probiotic interventions affecting leanness and obesity of subjects under a HFD, which are operated by modulating the tripartite relationship among the host, microbiota, and metabolites.

The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus.

Enterovirus 71 (EV71) has emerged as a neurological virus causing life-threatening diseases in young children and infants. Although EV71 vaccines in development have presented promising results in several clinical trials, the identified key antigen for improving the broad protective efficacy of EV71 vaccines has not been well investigated. In this report, we show that different multiplicities of infection (MOIs) of the B4(E59) virus significantly affect EV71 vaccine production in a serum-free microcarrier bioreactor system. The antigens produced from high MOIs of 10-1 and 10-2 exhibited higher yield and more infectious full particle (FP) contents in the EV71 vaccines than those produced with low MOIs of 10-4 and 10-6, leading to better cross-neutralizing efficacy. The C4(E36) neutralization results showed that only antisera raised from EV71 FPs provided substantial neutralizing titers against C4(E36), whereas empty particles (EPs) of EV71 conferred no efficacy. Competitive ELISA showed that anti-FP mainly binds to FPs and that 20% of antibodies bind to EPs, whereas most anti-EP binds EPs, with only 10% antibodies binding to FPs. VP1-adsorbed anti-FP lost most of the virus neutralization efficiency, suggesting that the VP1 subunit of FP is the major immunogenic antigen determining the ability of the EV71 vaccine to elicit cross-neutralizing antibodies against EV71 virus subtypes. These findings demonstrate that the high-MOI production approach is significantly correlated with FP productivity, thereby improving the cross-neutralization efficacy of an EV71 vaccine and providing the basis for a better vaccine design against widespread EV71 viruses.

Lack of mutagenicity, genotoxicity and developmental toxicity in safety assessment tests of Lactobacillus mali APS1.

Lactobacillus (L.) mali APS1 isolated from sugary kefir grains has been proven to affect energy and glucose homeostasis. However, without proper safety assessment it cannot be recommended as probiotics for human consumption. For genotoxicity, the Ames test showed no mutagenic effect of L. mali APS1 in the presence or absence of S9 mix metabolic activation. In-vitro mammalian chromosomal aberration test showed that the number of Chinese hamster ovary cells with abnormal chromosomes was <5% after L. mali APS1 treatment. Moreover, L. mali APS1 showed no risk of genotoxicity potential compared to the control. L. mali APS1 administration did not cause significant (p>0.05) changes in body weight, the number of reticulocytes, or in the occurrence percentage of micronucleus in Imprinting Control Region (ICR) mice. Based on the absence of maternal or fetal effects at any dosage level investigated, the teratogenicity could be defined as greater than 1,670 mg/kg b.w./day for maternal general toxicity and fetal development when L. mali APS1 was orally administered by gavage to pregnant SD rats during gestation days 6 to 15.

A combination of Lactobacillus mali APS1 and dieting improved the efficacy of obesity treatment via manipulating gut microbiome in mice.

The difficulty of long-term management has produced a high rate of failure for obesity patients. Therefore, improving the efficacy of current obesity treatment is a significant goal. We hypothesized that combining a probiotic Lactobacillus mali APS1 intervention with dieting could improve the efficacy of obesity and hepatic steatosis treatment compared to dieting alone. Mice were fed a high-fat diet for 6 weeks and then treated with: saline + normal diet and APS1 + normal diet (NDAPS1) for 3 weeks. NDAPS1 accelerated body weight loss and reduced caloric intake and fat accumulation. The fecal microbiome showed that accelerating weight loss by NDAPS1 resulted in restoring intestinal microbiota toward a pre-obese state, with alteration of specific changes in the obesity-associated bacteria. APS1 manipulated the gut microbiome's obesity-associated metabolites, followed by regulation of lipid metabolism, enhancement of energy expenditure and inhibition of appetite. The specific hepatic metabolites induced by the APS1-manipulated gut microbiome also contributed to the amelioration of hepatic steatosis. Our results highlighted a possible microbiome and metabolome that contributed to accelerating weight loss following treatment with a combination of APS1 and dieting and suggested that probiotics could serve as a potential therapy for modulating physiological function and downstream of the microbiota.

Sugary Kefir Strain Lactobacillus mali APS1 Ameliorated Hepatic Steatosis by Regulation of SIRT-1/Nrf-2 and Gut Microbiota in Rats.

SCOPE: Non-alcoholic fatty liver disease (NAFLD) is a common disease that is concomitant with obesity, resulting in increased mortality. To date, the efficiency of NAFLD treatment still needs to be improved. Therefore, we aimed to evaluate the effect of Lactobacillus mali APS1, which was isolated from sugary kefir, on hepatic steatosis in rats fed a high-fat diet (HFD). METHODS AND RESULTS: Sprague Dawley rats were fed a control diet, a HFD with saline, and a HFD with APS1 intervention by gavage daily for 12 weeks. The results showed that APS1 significantly reduced body weight and body weight gain in HFD-fed rats. APS1 reduced hepatic lipid accumulation by regulating SIRT-1/PGC-1α/SREBP-1 expression. Moreover, APS1 increased hepatic antioxidant activity by modulating Nrf-2/HO-1 expression. Notably, APS1 manipulated the gut microbiota, resulting in increasing proportions of the phylum Bacteroidetes/Firmicutes and reducing the abundance of specific NAFLD-associated bacteria. CONCLUSION: These results suggested that APS1 ameliorated hepatic steatosis by modulating lipid metabolism and antioxidant activity via manipulating specific NAFLD-associated gut microbiota in vivo.

Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10(7) TCID50/mL 10 days after infection when using an MOI of 10(-4). The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.

Squalene-adjuvanted H7N9 virus vaccine induces robust humoral immune response against H7N9 and H7N7 viruses.

Recent cases of avian influenza H7N9 have caused great concerns that virus may become transmittable between humans. It is imperative to develop an effective vaccine to fight against the pandemic potential of this H7N9 influenza virus to protect human from the disease. This study aims to investigate an optimized formulation for the development of H7N9 vaccines. Various doses of H7N9 inactivated whole or split-virus antigens (0.5, 1.5, or 3 µg based on hemagglutinin content) combined with squalene-based adjuvant (AddaVAX), aluminum hydroxide Al(OH)3 or without adjuvant were evaluated for the efficacy of H7N9 vaccine regiments in mice. With either H7N9 whole or split-virus based vaccines, AddaVAX-adjuvanted formulations were the most immunogenic in eliciting significant humoral immune response against H7N9 virus and exhibited strong cross-reactive response in hemagglutination inhibition (HAI) and viral-neutralization assays against H7N7 virus as well. In contrast, formulations with Al(OH)3 or without adjuvant were less immunogenic and elicited lower titers of HAI and microneutralization assays against both viruses. Dose-sparing experiments suggested that the formulation with as low as 0.004 µg of split or whole virus vaccine antigens together with 50% AddaVAX provided sufficient sero-protective HAI titers and achieved essential virus-neutralizing antibody titers against H7-subtype influenza viruses in mice. Protection experiments demonstrated that the formulation of 0.004 µg to 0.5 µg of split-virion vaccines with AddaVAX conferred full protection against viral challenge up to 100 LD50 of wild-type H7N9 virus, with 0% survival in placebo group. Taken together, our study demonstrates that squalene-based adjuvant can significantly enhance the protective efficacy of H7N9 virus vaccine and provides a useful strategy to confront the potential pandemic outbreaks of H7N9 virus.

Overcoming multidrug resistance of breast cancer cells by the micellar doxorubicin nanoparticles of mPEG-PCL-graft-cellulose.

The amphiphilic block copolymer methoxy-poly(ethylene glycol)-poly(epsilon-caprolactone) (mPEG-PCL) was grafted to 2-hydroxyethyl cellulose (HEC) to produce nano-sized micellar nanoparticles. The nanoparticles were loaded with anti-tumor drug, doxorubicin (DOX) and the size of the DOX-loaded nanoparticles were determined by dynamic light scattering (DLS) in aqueous solution to be from 197.4 to 230 nm. The nanoparticles subjected to co-culture with macrophage cells showed that these nanoparticles used as drug carrier are not recognized as foreign bodies. Overexpression of P-glycoprotein (P-gp) is an important factor in the development of multidrug resistance (MDR) in many cancer cells. In this study, Western blot and Rhodamine 123 were used to monitor the relative P-glycoprotein expression in human breast cancer cell lines MCF-7/WT and MCF-7/ADR. The endocytosis of the DOX-loaded nanoparticles by breast cancer cells is more efficient observed under a confocal laser scanning microscopy (CLSM) and a flow cytometry in MCF7/ADR cells, compared to the diffusion of the free drug into the cytoplasm of cells. Based on these findings, we concluded that the nanoparticles made from mPEG-PCL-g-cellulose were effective in overcoming P-gp efflux in MDR breast cancer cells.

Doxorubicin-loaded nanosized micelles of a star-shaped poly(ε-caprolactone)-polyphosphoester block co-polymer for treatment of human breast cancer.

Star-shaped co-polymers based on the backbone of poly(ε-caprolactone) were synthesized by a ring-opening reaction using pentaerythritol as initiator and Sn(Oct)(2) as catalyst. The star-shaped poly(ε-caprolactone) polymer was then chain extended with a terminal block of poly(ethyl ethylene phosphate) to form a copolymer, poly(ε-caprolactone)-poly(ethyl ethylene phosphate), when using the cyclic ethyl ethylene phosphate monomer. The amphiphilic block co-polymers can self-assemble into nanoscopic micelles with a mean diameter of 150 nm and a spherical shape. Additionally, the prepared micelles did not induce hemolysis and nitric oxide production in vitro based on nitric oxide, hemolytic tests and MTT assays. The hydrophobic micellar cores encapsulated doxorubicin (DOX) in an aqueous solution with a loading efficiency of 55.2%. The in vitro release of DOX from DOX-loaded micelles was pH dependent. DOX-loaded micelles present significantly enhanced cytotoxicity to both MCF-7/drug-sensitive and MCF-7/drug-resistant cells after second incubation. Moreover, results of confocal microscopy and flow cytometry of DOX-loaded micelles demonstrate the feasibility of this delivery system for effective therapy of drug-resistant tumours.

Nano-sized micelles of block copolymers of methoxy poly(ethylene glycol)-poly(epsilon-caprolactone)-graft-2-hydroxyethyl cellulose for doxorubicin delivery.

The amphiphilic block copolymers methoxy poly(ethylene glycol)-poly(epsilon-caprolactone) was grafted to 2-hydroxyethyl cellulose to produce the water-soluble copolymers. Doxorubicin loaded nanoparticles were prepared by dialysis method and the sizes of nanoparticles were determined by dynamic light scattering in solution and atomic force microscopes. As results the sizes were detected in a range of 197.4 to 340.7 nm. The in-vitro release of Dox was studied in phosphate and acetate buffered solution at 37 degrees C. The results showed that 43 and 53% of Dox remained after an incubation period of 7 days. The cytotoxicity of Dox loaded micelles was investigated in two different human MCF-7/wild type and MCF-7/Adriamycin drug resistant cells lines. The Dox-loaded micelles showed reduced cytotoxicity compared to free Dox in MCF-7/wild type and MCF-7/Adriamycin drug resistant cells.