Enteropathogenic Escherichia coli (EPEC), which belongs to the attaching and effacing diarrheagenic E. coli strains, is a major causative agent of life-threatening diarrhea in infants in developing countries. Most EPEC isolates correspond to certain O serotypes; however, many strains are non-typeable. Two EPEC strains, EPEC001 and EPEC080, which could not be serotyped during routine detection, were isolated. In this study, we conducted an in-depth characterization of their putative O-antigen gene clusters (O-AGCs) and also performed constructed mutagenesis of the O-AGCs for functional analysis of O-antigen (OAg) synthesis. Sequence analysis revealed that the occurrence of O-AGCs in EPEC001 and E. coli O132 may be mediated by recombination between them, and EPEC080 and E. coli O2/O50 might acquire each O-AGC from uncommon ancestors. We also indicated that OAg-knockout bacteria were highly adhesive in vitro, except for the EPEC001 wzy derivative, whose adherent capability was less than that of its wild-type strain, providing direct evidence that OAg plays a key role in EPEC pathogenesis. Together, we identified two EPEC O serotypes in silico and experimentally, and we also studied the adherent capabilities of their OAgs, which highlighted the fundamental and pathogenic role of OAg in EPEC.

Sepsis is an acute inflammatory response that leads to life-threatening complications if not quickly and adequately treated. Cytolysin, hemolysin, and pneumolysin are toxins produced by gram-positive bacteria and are responsible for resistance to antimicrobial drugs, cause virulence and lead to sepsis. This work assessed the effects of aloe-emodin (AE) and photodynamic therapy (PDT) on sepsis-associated gram-positive bacterial toxins. Standard and antibiotic-resistant Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumonia bacterial strains were cultured in the dark with varying AE concentrations and later irradiated with 72 J/cm^-2 light. Colony and biofilm formation was determined. CCK-8, Griess reagent reaction, and ELISA assays were done on bacteria-infected RAW264.7 cells to determine the cell viability, NO, and IL-1β and IL-6 pro-inflammatory cytokines responses, respectively. Hemolysis and western blot assays were done to determine the effect of treatment on hemolysis activity and sepsis-associated toxins expressions. AE-mediated PDT reduced bacterial survival in a dose-dependent manner with 32 ㎍/ml of AE almost eliminating their survival. Cell proliferation, NO, IL-1β, and IL-6 cytokines production were also significantly downregulated. Further, the hemolytic activities and expressions of cytolysin, hemolysin, and pneumolysin were significantly reduced following AE-mediated PDT. In conclusion, combined use of AE and light (435 ± 10 nm) inactivates MRSA, S. aureus (ATCC 29213), S. pneumoniae (ATCC 49619), MDR-S. pneumoniae, E. faecalis (ATCC 29212), and VRE (ATCC 51299) in an AE-dose dependent manner. AE and light are also effective in reducing biofilm formations, suppressing pro-inflammatory cytokines, hemolytic activities, and inhibiting the expressions of toxins that cause sepsis.

Two gram-negative, catalase-positive, strictly aerobic, and white colony-forming bacteria, strains H242^T and B156^T, were isolated from soil in South Korea. Cells of strain H242^T were oxidase-positive and non-motile short rods, while those of strain B156^T were oxidase-negative and long non-motile rods. Ubiquinone-8 was identified as the sole isoprenoid quinone in both strains. C_16:0, cyclo-C_17:0, andsummed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were identified in both strains as the major cellular fatty acids and polar lipids, respectively. The DNA G+C contents of strains H242^T and B156^T were 69.4 mol% and 69.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA and 92 concatenated core gene sequences revealed that strains H242^T and B156^T formed distinct phylogenic lineages from other Ramlibacter type strains. The DNA-DNA hybridization (DDH) value between strains H242^T and B156^T was 24.6%. Strains H242^T and B156^T were most closely related to Ramlibacter ginsenosidimutans BXN5-27^T and Ramlibacter monticola G-3-2^T with 98.4% and 98.6% 16S rRNA gene sequence similarities, respectively. Digital DDH values between strain H242^T and R. ginsenosidimutans and between strain B156^T and R. monticola were 23.5% and 26.1%, respectively. Phenotypic, chemotaxonomic, and molecular analyses indicated that strains H242^T and B156^T represent two novel species of the genus Ramlibacter, for which the names Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., respectively, are proposed. The type strains of R. terrae and R. montanisoli are H242^T (=KACC 21667^T=JCM 33922^T) and B156^T (=KACC 21665^T=JCM 33920^T), respectively.

Cold-adapted plant growth-promoting bacteria (PGPB) with multiple functions are an important resource for microbial fertilizers with low-temperature application. In this study, culturable cold-adapted PGPB strains with nitrogen fixation and phosphorus solubilization abilities were isolated. They were screened from root and rhizosphere of four dominant grass species in nondegraded alpine grasslands of the Qilian Mountains, China. Their other growth-promoting characteristics, including secretion of indole-3-acetic acid (IAA), production of siderophores and ACC deaminase, and antifungal activity, were further studied by qualitative and quantitative methods. In addition, whether the PGPB strains could still exert plant growth-promoting activity at 4℃ was verified. The results showed that 67 isolates could maintain one or more growth-promoting traits at 4℃, and these isolates were defined as cold-adapted PGPB. They were divided into 8 genera by 16S rRNA gene sequencing and phylogenetic analysis, of which Pseudomonas (64.2%) and Serratia (13.4%) were the common dominant genera, and a few specific genera varied among the plant species. A test-tube culture showed that inoculation of Elymus nutans seedlings with cold-adapted PGPB possessing different functional characteristics had a significant growth-promoting effect under controlled low-temperature conditions, including the development of the roots and aboveground parts. Pearson correlation analysis revealed that different growth-promoting characteristics made different contributions to the development of the roots and aboveground parts. These cold-adapted PGPB can be used as excellent strain resources suitable for the near-natural restoration of degraded alpine grasslands or agriculture stock production in cold areas.

Members of the genus Bacillus are known to play an important role in promoting plant growth and protecting plants against phytopathogenic microorganisms. In this study, 21 isolates of Bacillus spp. were obtained from the root micro-ecosystem of Suaeda glauca. Analysis of the 16S rRNA genes indicated that the isolates belong to the species Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus aryabhattai and Brevibacterium frigoritolerans. One of the interesting findings of this study is that the four strains B1, B5, B16 and B21 are dominant in rhizosphere soil. Based on gyrA, gyrB, and rpoB gene analyses, B1, B5, and B21 were identified as B. amyloliquefaciens and B16 was identified as B. velezensis. Estimation of antifungal activity showed that the isolate B1 had a significant inhibitory effect on Fusarium verticillioides, B5 and B16 on Colletotrichum capsici (syd.) Butl, and B21 on Rhizoctonia cerealis van der Hoeven. The four strains grew well in medium with 1-10% NaCl, a pH value of 5-8, and promoted the growth of Arabidopsis thaliana. Our results indicate that these strains may be promising agents for the biocontrol and promotion of plant growth and further study of the relevant bacteria will provide a useful reference for the development of microbial resources.

This study was carried out to explore a non-chemical strategy for enhancing productivity by employing some antagonistic rhizobacteria. One hundred eighteen bacterial isolates were obtained from the rhizospheric zone of various crop fields of Gangwon-do, Korea, and screened for antifungal activity against Fusarium wilt (Fusarium oxysporum f. sp. lactucae) in lettuce crop under in vitro and in vivo conditions. In broth-based dual culture assay, fourteen bacterial isolates showed significant inhibition of mycelial growth of F. oxysporium f. sp. lactucae. All of the antagonistic isolates were further characterized for the antagonistic traits under in vitro conditions. The isolates were identified on the basis of biochemical characteristics and confirmed at their species level by 16S rRNA gene sequencing analysis. Arthrobacter sulfonivorans, Bacillus siamensis, Bacillus amyloliquefaciens, Pseudomonas proteolytica, four Paenibacillus peoriae strains, and Bacillus subtilis were identified from the biochemical characterization and 16S rRNA gene sequencing analysis. The isolates EN21 and EN23 showed significant decrease in disease severity on lettuce compared to infected control and other bacterial treatments under greenhouse conditions. Two bacterial isolates, EN4 and EN21, were evaluated to assess their disease reduction and growth promotion in lettuce in field conditions. The consortium of EN4 and EN21 showed significant enhancement of growth on lettuce by suppressing disease caused by F. oxysporum f. sp. lactucae respectively. This study clearly indicates that the promising isolates, EN4 (P. proteolytica) and EN21 (Bacillus siamensis), can be commercialized and used as biofertilizer and/or biopesticide for sustainable crop production.

Rubus coreanus Miquel (bokbunja), Korean black raspberry, is known to possess various phytochemicals that exert antioxidative, anti-inflammatory, and anti-cancer effects. However, most studies on Rubus coreanus Miquel have been performed with the solvent extracts and/or a single component to demonstrate the efficacy, while studies evaluating the effect of the whole fructus of Rubus coreanus Miquel are limited. In this study, therefore, we employed the isoproterenol (IPN)-induced myocardial infarction model and investigated the effect of freeze-dried powder of Rubus coreanus Miquel (RCP) on oxidative stress and prevention of organ damage. Oral administration of RCP reduced the level of toxicity markers, alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) without affecting body weight and diet intake. The oxidative stress marker glutathione (GSH) increased about 45% and malonaldehyde (MDA) decreased about 27% compared to the IPN group with RCP-H (3%) administration. By histological analysis, IPN induced significant myocardial damage in the heart and vascular injury in the liver, and RCP administration ameliorated the damages in a dose-dependent manner. Taken together, RCP activated the antioxidant system leading to prevention of damage to organs by IPN in rats, making it possible to expect beneficial efficacies by consuming the whole fructus of Rubus coreanus Miquel.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

With changes in human dietary patterns, the proportion of high-fat and high-cholesterol foods in the daily diet has increased. As a result, the incidence rate of cholelithiasis is increasing rapidly. Many studies have reported on the crucial role that the intestinal microflora plays in the progression of gallstones. Although the whole herb of Lysimachia christinae, a traditional Chinese medicine, has long been extensively used as a remedy for cholelithiasis in China, its effects on the intestinal microflora remain unknown. Hence, in this study, we investigated the ability of the aqueous extract of L. christinae (LAE) to prevent cholesterol gallstones (CGSs) in model animals by affecting the intestinal microflora. The effects of LAE on body weight, serum lipid profile, visceral organ indexes, and histomorphology were studied in male C57BL/6J mice, which were induced by a lithogenic diet. After the 8-week study, CGSs formation was greatly reduced after LAE treatment. LAE also reduced body weight gain and hyperlipidemia and restored the histomorphological changes. Moreover, the intestinal microflora exhibited significant variation. In the model group fed the lithogenic diet, the abundances of the genera unclassified Porphyromonadaceae, Lactobacillus and Alloprevotella decreased, but in contrast, Akkermansia dramatically increased compared with the control check group, which was fed a normal diet; the administration of LAE reversed these changes. These results imply that L. christinae can be considered an efficient therapy for eliminating CGSs induced by a high-fat and high-cholesterol diet, which may be achieved by influencing the intestinal microflora.

Clinical and preclinical studies have reported that Lactobacillus gasseri BNR17, a probiotic bacterial strain isolated from human breast milk, reduces body weight and white adipose tissue volume. In order to further explore the actions of L. gasseri BNR17, we investigated the anti-menopausal effects of L. gasseri BNR17 in an ovariectomized (OVX) rat model. The serum alanine aminotransferase levels of the rats in the OVX-BNR17 group were lower than those of the rats in the OVX-vehicle only (OVX-Veh) group. Upon administration of L. gasseri BNR17 after ovariectomy, calcitonin and Serotonin 2A levels increased significantly, whereas serum osteocalcin levels showed a decreasing tendency. Compared to the rats in the OVX-Veh group, those in the OVX-BNR17 group showed lower urine deoxypyridinoline levels, lower pain sensitivity, and improved vaginal cornification. Furthermore, L. gasseri BNR17 administration increased bone mineral density in the rats with OVX-induced femoral bone loss. These results suggest that L. gasseri BNR17 administration could alleviate menopausal symptoms, indicating that this bacterium could be a good functional probiotic for managing the health of older women.

There are a growing number of reports of hospital-acquired infections caused by pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). Many plant products are now being used as a natural means of exploring antimicrobial agents against different types of human pathogenic bacteria. In this research, we sought to isolate and identify an active molecule from Sedum takesimense that has possible antibacterial activity against various clinical isolates of MRSA. NMR analysis revealed that the structure of the HPLC-purified compound was 1,2,4,6-tetra-O-galloyl-glucose. The minimum inhibitory concentration (MIC) of different extract fractions against numerous pathogenic bacteria was determined, and the actively purified compound has potent antibacterial activity against multidrug-resistant pathogenic bacteria, i.e., MRSA and its clinical isolates. In addition, the combination of the active compound and β-lactam antibiotics (e.g., oxacillin) demonstrated synergistic action against MRSA, with a fractional inhibitory concentration (FIC) index of 0.281. The current research revealed an alternative approach to combating pathogenesis caused by multi-drug resistant bacteria using plant materials. Furthermore, using a combination approach in which the active plant-derived compound is combined with antibiotics has proved to be a successful way of destroying pathogens synergistically.

Modified Renshen Wumei decoction (MRWD), a famous traditional Chinese medicine, is widely used for treating persistent diarrhea. However, as the mechanism by which MRWD regulates diarrhea remains unknown, we examined the protective effects of MRWD on intestinal barrier integrity in a diarrhea model. In total, 48 male rats were randomly distributed to four treatment groups: the blank group (CK group), model group (MC group), Medilac-Vita group (MV group) and Chinese herb group (MRWD group). After a 21-day experiment, serum and colon samples were assessed. The diarrhea index, pathological examination findings and change in ᴅ-lactate and diamine oxidase (DAO) contents illustrated that the induction of diarrhea caused intestinal injury, which was ameliorated by MV and MRWD infusion. Metabolomics analysis identified several metabolites in the serum. Some critical metabolites, such as phosphoric acid, taurine, cortisone, leukotriene B4 and calcitriol, were found to be significantly elevated by MRWD infusion. Importantly, these differences correlated with mineral absorption and metabolism and peroxisome proliferator-activated receptor (PPAR) pathways. Moreover, it significantly increased the expression levels of TLR4, MyD88 and p-NF-κB p65 proteins and the contents of IL-1 and TNF-α, while the expression levels of occludin, claudin-1 and ZO-1 proteins decreased. These deleterious effects were significantly alleviated by MV and MRWD infusion. Our findings indicate that MRWD infusion helps alleviate diarrhea, possibly by maintaining electrolyte homeostasis, improving the intestinal barrier integrity, and inhibiting the TLR4/NF-κB axis.

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

Microbially induced calcium carbonate precipitation (MICP) has recently become an intelligent and environmentally friendly method for repairing cracks in concrete. To improve on this ability of microbial materials concrete repair, we applied random mutagenesis and optimization of mineralization conditions to improve the quantity and crystal form of microbially precipitated calcium carbonate. Sporosarcina pasteurii ATCC 11859 was used as the starting strain to obtain the mutant with high urease activity by atmospheric and room temperature plasma (ARTP) mutagenesis. Next, we investigated the optimal biomineralization conditions and precipitation crystal form using Plackett-Burman experimental design and response surface methodology (RSM). Biomineralization with 0.73 mol/l calcium chloride, 45 g/l urea, reaction temperature of 45℃, and reaction time of 22 h, significantly increased the amount of precipitated calcium carbonate, which was deposited in the form of calcite crystals. Finally, the repair of concrete using the optimized biomineralization process was evaluated. A comparison of water absorption and adhesion of concrete specimens before and after repairs showed that concrete cracks and surface defects could be efficiently repaired. This study provides a new method to engineer biocementing material for concrete repair.

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.