Obesity, a major healthcare problem worldwide, induces metabolic endotoxemia through the gut translocation of lipopolysaccharides (LPS), a major cell wall component of Gram-negative bacteria, causing ...a chronic inflammatory state. A combination of several probiotics including Lactobacillus acidophilus 5 (LA5), a potent lactic acid-producing bacterium, has previously been shown to attenuate obesity. However, data on the correlation between a single administration of LA5 versus microbiota alteration might be helpful for the probiotic adjustment. LA5 was administered daily together with a high-fat diet (HFD) for 8 weeks in mice. Furthermore, the condition media of LA5 was also tested in a hepatocyte cell-line (HepG2 cells). Accordingly, LA5 attenuated obesity in mice as demonstrated by weight reduction, regional fat accumulation, lipidemia, liver injury (liver weight, lipid compositions, and liver enzyme), gut permeability defect, endotoxemia, and serum cytokines. Unsurprisingly, LA5 improved these parameters and acidified fecal pH leads to the attenuation of fecal dysbiosis. The fecal microbiome analysis in obese mice with or without LA5 indicated; (i) decreased Bacteroidetes (Gram-negative anaerobes that predominate in non-healthy conditions), (ii) reduced total fecal Gram-negative bacterial burdens (the sources of gut LPS), (iii) enhanced Firmicutes (Gram-positive bacteria with potential benefits) and (iv) increased Verrucomycobia, especially Akkermansia muciniphila, a bacterium with the anti-obesity property. With LA5 administration, A. muciniphila in the colon were more than 2,000 folds higher than the regular diet mice as determined by 16S rRNA. Besides, LA5 produced anti-inflammatory molecules with a similar molecular weight to LPS that reduced cytokine production in LPS-activated HepG2 cells. In conclusion, LA5 attenuated obesity through (i) gut dysbiosis attenuation, partly through the promotion of A. muciniphila (probiotics with the difficulty in preparation processes), (ii) reduced endotoxemia, and (iii) possibly decreased liver injury by producing the anti-inflammatory molecules.
Gut fungi may influence the course of Clostridium difficile infection either positively or negatively for the host. Fungi are not prominent in the mouse gut, and C. albicans, the major human ...gastrointestinal commensal yeast, is in low abundance or absent in mice. Bifidobacterium is one of the probiotics that may attenuate the severity of C. difficile infection. Inflammatory synergy between C. albicans and C. difficile, in gut, may provide a state that more closely resembles human infection and be more suitable for testing probiotic effects. We performed fecal mycobiota analysis and administered C. albicans at 1 day prior to C. difficile dosing. Fecal eukaryotic 18S rDNA analysis demonstrated the presence of Ascomycota, specifically, Candida spp., after oral antibiotics, despite negative fecal fungal culture. C. albicans administration enhanced the severity of the C. difficile infection model as determined by mortality rate, weight loss, gut leakage (FITC-dextran assay), and serum and intestinal tissue cytokines. This occurred without increased fecal C. difficile or bacteremia, in comparison with C. difficile gavage alone. Candida lysate with C. difficile increased IL-8 production from HT-29 and Caco-2 human intestinal epithelial cell-lines. Bifidobacterium attenuated the disease severity of the C. difficile plus Candida model. The reduced severity was associated with decreased Candida burdens in feces. In conclusion, gut C. albicans worsened C. difficile infection, possibly through exacerbation of inflammation. Hence, a mouse model of Clostridium difficile infection with C. albicans present in the gut may better model the human patient condition. Gut fungal mycobiome investigation in patients with C. difficile is warranted and may suggest therapeutic targets.
Northeastern Thailand relies on agriculture as a major economic activity, and has used high levels of agrochemicals due to low facility, and salty sandy soil. To support soil recovery and sustainable ...agriculture, local farmers have used organic fertilizers from farmed animal feces. However, knowledge about these animal fecal manures remains minimal restricting their optimal use. Specifically, while bacteria are important for soil and plant growth, an abundance and a diversity of bacterial composition in these animal fecal manures have not been reported to allow selection and adjustment for a more effective organic fertilizer. This study thereby utilized metagenomics combined with 16S rRNA gene quantitative PCR (qPCR) and sequencing to analyze quantitative microbiota profiles in association with nutrients (N, P, K), organic matters, and the other physiochemical properties, of the commonly used earthworm manure and other manures from livestock animals (including breed and feeding diet variations) in the region. Unlike the other manures, the earthworm manure demonstrated more favorable nutrient profiles and physiochemical properties for forming fertile soil. Despite low total microbial biomass, the microbiota were enriched with maximal OTUs and Chao richness, and no plant pathogenic bacteria were found based on the VFDB database. The microbial metabolic potentials supported functions to promote crop growth, such as C, N and P cyclings, xenobiotic degradation, and synthesis of bioactive compounds. Pearson's correlation analyses indicated that the quantitative microbiota of the earthworm manure were clustered in the same direction as N, and conductivity, salinity, and water content were essential to control the microbiota of animal manures.
An outbreak of citrus greening or Huanglongbing disease bacteria occurs in many areas. We sampled and identified an ongoing ~year 2020 orange tree endemic in northern Thailand as
Candidatus
...Liberibacter asiaticus. We thereby developed a plant greening disease (
C
. Liberibacter asiaticus) detection assay using simple alkaline heat DNA lysis and loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB), and evaluated the developed assay for its feasibility as point-of-care detection on 65 plant leaf samples with 100–1×10
4
copies of
C
. Liberibacter asiaticus or mocked injection compared with commercial DNA lysis kit and PCR-GE. Our assay is sensitive to 5–8.9 copies of
omp
(equaling 0.0056–0.01 fg) compatible with PCR-GE limit of detection. This ultra sensitive limit of detection could allow the disease detection before clinical apparent state of disease when
C
. Liberibacter asiaticus infection number is few, i.e. fewer than 100 copies of
C
. Liberibacter asiaticus. The assay is also specific with 6 degenerate primers targeting every strain of
C
. Liberibacter asiaticus
omp
from GenBank database, rapid (40 min total assay time), inexpensive (~2–3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay accuracy (93.85–100% accuracy, 100% specificity, and 89.74–100% sensitivity) to bacterial DNA extraction by a commercial kit followed by PCR and gel electrophoresis (92.31% accuracy, 100% specificity, and 87.18% sensitivity) based on the real sample tests. Hence, the technique could be used in local or laboratory resource-restricted settings. The test result could be read by naked eyes through the color change from violet (negative) to sky blue (positive) for a
C
. Liberibacter asiaticus-infected specimen. Furthermore, this assay uses safe chemical reagents and, thus, is safe for the users.
is abundant in the human gut mycobiota but this species does not colonize the mouse gastrointestinal tract. C. albicans administration in dextran-sulfate solution (DSS) induced-colitis mouse model ...(DSS+Candida) might resemble more to human condition, therefore, a DSS colitis model with Candida administration was studied; first, to test the influence of fungi in DSS model and second, to test the efficacy of Lactobacillus rhamnosus L34. We demonstrated serum (1→3)-β-D-glucan (BG) elevation in patients with IBD and endoscopic moderate colitis in clinical remission, supporting the possible influence of gut fungi toward IBD in human. Then, in mouse model, Candida gavage was found to worsen the DSS model indicated by higher mortality rate, more severe colon histology and enhanced gut-leakage (FITC-dextran assay, endotoxemia, serum BG and blood bacterial burdens) but did not affect weight loss and diarrhea. DSS+Candida induced higher pro-inflammatory cytokines both in blood and in intestinal tissue. Worsened systemic pro-inflammatory cytokine responses in DSS+Candida compared with DSS alone was possibly due to the more severe translocation of LPS, BG and bacteria (not fungemia) from gut into systemic circulation. Interestingly, bacteremia from Pseudomonas aeruginosa was more frequently isolated from DSS+Candida than DSS alone. In parallel, P. aeruginosa was also isolated from fecal culture in most of the mice in DSS+Candida group supported by prominent Gammaproteobacteria in fecal microbioata analysis. However, L. rhamnosus L34 attenuated both DSS+Candida and DSS model through the attenuation of gut local inflammation (cytokines and histology), gut-leakage severity, fecal dysbiosis (culture method and microbiome analysis) and systemic inflammation (serum cytokines). In conclusion, gut Candida in DSS model induced fecal bacterial dysbiosis and enhanced leaky-gut induced bacteremia. Probiotic treatment strategy aiming to reduce gut-fungi and fecal dysbiosis could attenuate disease severity. Investigation on gut fungi in patients with IBD is highly interesting.
Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus ...rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-β-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02-2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.
Macrophages are responsible for the recognition of pathogen molecules. The downstream signalling of the innate immune responses against pathogen molecules, lipopolysaccharide (LPS) and ...(1→3)-β-D-glucan (BG), and the adaptive immune response to antibodies, Fc gamma receptor (FcgR), is spleen tyrosine kinase (Syk). Because pathogen molecules and antibodies could be presented in lupus, impact of Syk and macrophages in lupus is explored. FcgR-IIb deficient (FcgRIIb-/-) mice, a model of inhibitory signalling loss, at 40 weeks old, but not pristane mice (a chemical induction lupus model) demonstrated spontaneous elevation of LPS and BG in serum from gut translocation despite the similarity in faecal microbiome analysis. Syk abundance in FcgRIIb–/– mice was higher than in pristane mice, possibly due to several Syk activators (anti-dsDNA, LPS and BG), and Syk inhibitor–attenuated proteinuria and serum cytokines only in FcgRIIb–/– mice. In addition, LPS + BG enhanced the expression of activating FcgRs, NF-κB and Syk, together with supernatant TNF-α predominantly in FcgRIIb–/– compared to wild-type macrophages. The inhibitors against Dectin-1, Syk and nuclear factor kappa B, but not anti-Raf-1, reduced supernatant TNF-α in LPS+BG-activated macrophages, implying Syk-dependent signalling. The pathogen molecules enhanced activating-FcgRs, without inhibition, through Syk, a shared downstream innate and adaptive signalling, is responsible for the hyper-responsiveness in FcgRIIb–/– macrophages. In conclusion, Syk inhibitor attenuated inflammation in FcgRIIb–/– but not in pristane mice, implying the influence of a lupus genetic background in treatment modalities.
The influence of gut-leakage or gut-microbiota upon lupus progression was explored in 2 lupus mouse models. Pristane, administered in 4-wk-old wild-type (WT) female mice, induced lupus ...characteristics at 24-wk-old similar to the lupus-onset in FcGRIIb-/- mice. Gut-microbiota alteration was induced by co-housing together with the gavage of feces from 40-wk-old FcGRIIb-/- mice (symptomatic lupus). On the other hand, gut-leakage was induced by dextran sulfate solution (DSS). DSS and gut-microbiota alteration induced high serum anti-dsDNA immunoglobulin (Ig) as early as 30 days post-DSS only in FcGRIIb-/- mice. DSS, but not gut-microbiota alteration, enhanced lupus characteristics (serum creatinine and proteinuria) in both lupus models (but not in WT) at 60 days post-DSS. Indeed, DSS induced the translocation of molecular components of gut-pathogens as determined by bacterial burdens in mesenteric lymph node (MLN), endotoxemia (gut-bacterial molecule) and serum (1→3)-β-D-glucan (BG) (gut-fungal molecule) as early as 15 days post-DSS together with enhanced MLN apoptosis in both WT and lupus mice. However, DSS induced spleen apoptosis in FcGRIIb-/- and WT mice at 30 and 60 days post-DSS, respectively, suggesting the higher impact of gut-leakage against spleen of lupus mice. In addition, macrophages preconditioning with LPS plus BG were susceptible to starvation-induced apoptosis, predominantly in FcGRIIb-/- cell, implying the influence of gut-leakage upon cell stress. In summary, gut-leakage induced gut-translocation of organismal-molecules then enhanced the susceptibility of stress-induced apoptosis, predominantly in lupus. Subsequently, the higher burdens of apoptosis in lupus mice increased anti-dsDNA Ig and worsen lupus severity through immune complex deposition. Hence, therapeutic strategies addressing gut-leakage in lupus are interesting.
Abstract
A symbiosis of bacterial community (sometimes called microbiota) play essential roles in developmental life cycle and health of coral, starting since a larva. For examples, coral bacterial ...holobionts function nitrogen fixation, carbon supply, sulfur cycling and antibiotic production. Yet, a study of the dynamic of bacteria associated coral larvae development is complicated owning to a vast diversity and culturable difficulty of bacteria; hence this type of study remains unexplored for
Acropora humilis
larvae in Thai sea. This study represented the first to utilize 16S rRNA gene sequencing to describe the timely bacterial compositions during successfully cultured and reared
A. humilis
larval transformation in aquaculture (gametes were collected from Sattahip Bay, Chonburi province, Thailand), from gamete spawning (0 h) and fertilization stage (1 h), to embryonic cleavage (8 h), round cell development (28, 39 and 41 h), and planula formation (48 h). The sequencing results as estimated by Good’s coverage at genus level covered 99.65 ± 0.24% of total bacteria. While core phyla of bacteria were observed (Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes), changes in bacterial population structures and differential predominant core bacterial orders were denoted for each larval developmental stage, from fertilization to embryonic cleavage and subsequently from the embryonic cleavage to round cell development (
P
= 0.007). For instances,
Pseudoalteromonas
and Oceanospirillales were found prevalent at 8 h, and Rhizobiales were at 48 h. The bacterial population structures from the round cell stage, particularly at 41 h, showed gradual drift towards those of the planula formation stage, suggesting microbial selection. Overall, this study provides preliminary insights into the dynamics of bacterial community and their potentially functional association (estimated from the bacterial compositions) during the developmental embryonic
A. humilis
in a cultivation system in Southeast Asia region.
Staphylococcus aureus strains carrying enterotoxin A gene (sea) causes food poisoning and cannot be distinguished from non-pathogenic strains by the culture method. Here, we developed a rapid, ...specific and sensitive visual detection of sea using loop-mediated isothermal amplification (LAMP) combined with nanogold probe (AuNP) or styryl dye (STR). LAMP-AuNP and LAMP-STR can detect as low as 9.7 fg (3.2 sea copies) and 7.2 sea copies, respectively, which were lower than PCR (97 fg or 32 sea copies). The excellent performance of these new assays was demonstrated in food samples using crude DNA lysates. While the culture method detected 10
CFU/g in ground pork and 10 CFU/mL in milk in 5-7 days, LAMP-AuNP could detect down to 10 CFU/g for both samples in 27 minutes. Analyzing 80 pork and milk samples revealed that the LAMP-AuNP showed 100% sensitivity, 97-100% specificity and 97.5-100% accuracy, which were superior to the culture method, and comparable to PCR but without requirement of a thermal cycler. Furthermore, our LAMP-AuNP detect sea at a range below the food safety control (<100 CFU/g). The LAMP-STR quantitated sea in 10-1,000 CFU (7.2-720 copies). Our crude DNA lysis combined with LAMP-AuNP/STR present effective point-of-care detection and facilitate appropriate control strategies.
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