Search results for: lactobacillus plantarum

Authors: Anuj Tyagi, Balwinder Singh, Naveen Kumar B. T., Niraj K. Singh

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Characterization of diverse microbial communities in specific environment plays a crucial role in the better understanding of their functional relationship with the ecosystem. It is now well established that gut microbiome of fish is not the simple replication of microbiota of surrounding local habitat, and extensive species, dietary, physiological and metabolic variations in fishes may have a significant impact on its composition. Moreover, overuse of antibiotics in human, veterinary and aquaculture medicine has led to rapid emergence and propagation of antibiotic resistance genes (ARGs) in the aquatic environment. Microbial communities harboring specific ARGs not only get a preferential edge during selective antibiotic exposure but also possess the significant risk of ARGs transfer to other non-resistance bacteria within the confined environments. This phenomenon may lead to the emergence of habitat-specific microbial resistomes and subsequent emergence of virulent antibiotic-resistant pathogens with severe fish and consumer health consequences. In this study, gut microbiota of freshwater carp (Labeo rohita) was investigated by shotgun metagenomics to understand its taxonomic composition and functional capabilities. Metagenomic DNA, extracted from the fish gut, was subjected to sequencing on Illumina NextSeq to generate paired-end (PE) 2 x 150 bp sequencing reads. After the QC of raw sequencing data by Trimmomatic, taxonomic analysis by Kraken2 taxonomic sequence classification system revealed the presence of 36 phyla, 326 families and 985 genera in the fish gut microbiome. At phylum level, Proteobacteria accounted for more than three-fourths of total bacterial populations followed by Actinobacteria (14%) and Cyanobacteria (3%). Commonly used probiotic bacteria (Bacillus, Lactobacillus, Streptococcus, and Lactococcus) were found to be very less prevalent in fish gut. After sequencing data assembly by MEGAHIT v1.1.2 assembler and PROKKA automated analysis pipeline, pathway analysis revealed the presence of 1,608 Metacyc pathways in the fish gut microbiome. Biosynthesis pathways were found to be the most dominant (51%) followed by degradation (39%), energy-metabolism (4%) and fermentation (2%). Almost one-third (33%) of biosynthesis pathways were involved in the synthesis of secondary metabolites. Metabolic pathways for the biosynthesis of 35 antibiotic types were also present, and these accounted for 5% of overall metabolic pathways in the fish gut microbiome. Fifty-one different types of antibiotic resistance genes (ARGs) belonging to 15 antimicrobial resistance (AMR) gene families and conferring resistance against 24 antibiotic types were detected in fish gut. More than 90% ARGs in fish gut microbiome were against beta-lactams (penicillins, cephalosporins, penems, and monobactams). Resistance against tetracycline, macrolides, fluoroquinolones, and phenicols ranged from 0.7% to 1.3%. Some of the ARGs for multi-drug resistance were also found to be located on sequences of plasmid origin. The presence of pathogenic bacteria and ARGs on plasmid sequences suggested the potential risk due to horizontal gene transfer in the confined gut environment.

Keywords: antibiotic resistance, fish gut, metabolic pathways, microbial diversity

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Authors: Sandhya Parameswaran, Prajakta Dhuri

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Despite great improvements in health care, the world oral health report states that dental problems still persist, particularly among underprivileged groups in both developing and developed countries. Dental caries and periodontal diseases are identified as the most important oral health problems globally. Acidic foods and beverages can affect natural teeth, and chronic exposure often leads to the development of dental erosion, abrasion, and decay. In recent years, there has been an increased interest toward essential oils. These are secondary metabolites and possess antibacterial, antifungal and antioxidant properties. Essential oils are volatile and chemically unstable in the presence of air, light, moisture and high temperature. Hence many novel methods like a liposomal encapsulation of oils have been introduced to enhance the stability and bioavailability. This research paper focuses on two essential oils, clove and cinnamon oil. Clove oil was obtained from Syzygium aromaticum Linn using clavengers apparatus. It contains eugenol and β caryophyllene. Cinnamon oil, from the barks of Cinnamomum cassia, contains cinnamaldehyde, The objective of the current research was to develop a liposomal carrier system containing clove and cinnamon oil and study their synergistic activity against dental pathogens when formulated as a gel. Methodology: The essential oil were first tested for their antimicrobial activity against dental pathogens, Lactobacillus acidophillus (MTCC No. 10307, MRS broth) and Streptococcus Mutans (MTCC No .890, Brain Heart Infusion agar). The oils were analysed by UV spectroscopy for eugenol and cinnamaldehyde content. Standard eugenol was linear between 5ppm to 25ppm at 282nm and standard cinnamaldehde from 1ppm to 5pmm at 284nm. The concentration of eugenol in clove oil was found to be 62.65 % w/w, and that of cinnamaldehyde was found to be 5.15%s w/w. The oils were then formulated into liposomes. Liposomes were prepared by thin film hydration method using Phospholipid, Cholesterol, and other oils dissolved in a chloroform methanol (3:1) mixture. The organic solvent was evaporated in a rotary evaporator above lipid transition temperature. The film was hydrated with phosphate buffer (pH 5.5).The various batches of liposomes were characterized and compared for their size, loading rate, encapsulation efficiency and morphology. The prepared liposomes when evaluated for entrapment efficiency showed 65% entrapment for clove and 85% for cinnamon oil. They were also tested for their antimicrobial activity against dental pathogens and their synergistic activity studied. Based on the activity and the entrapment efficiency the amount of liposomes required to prepare 1gm of the gel was calculated. The gel was prepared using a simple ointment base and contained 0.56% of cinnamon and clove liposomes. A simultaneous method of analysis for eugenol and cinnamaldehyde.was then developed using HPLC. The prepared gels were then studied for their stability as per ICH guidelines. Conclusion: It was found that liposomes exhibited spherical shaped vesicles and protected the essential oil from degradation. Liposomes, therefore, constitute a suitable system for encapsulation of volatile, unstable essential oil constituents.

Keywords: cinnamon oil, clove oil, dental caries, liposomes

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Authors: Hye-Hyun Lee, Hyo-Kon Chun, Kyung-Hee Kim Kim, Mi-Hee Kim, Saet-Byul Chu, Sang-Jong Lee, Seung-Hyeop Lee, Seung-Won Yi

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Some chemicals such as malachite green, methylene blue, and copper sulfate had been used frequently as disinfectants controlling fungal infection in aquaculture. However, their carcinogenicity, mutagenicity and teratogenicity were reported in mammals. After their accumulation in food fish and its consumers was confirmed, concerns about public health has resulted in enhanced monitoring and increased demand for eco-friendly treatments. Therefore, this study aimed to evaluate safety to fish and efficacy of sulfur solution processed by effective microorganisms (EM-PSS) against Saprolegnia parasitica, for use of a potential aquatic fungicidal disinfectant. The natural sulfur purchased from Kawah Ijen volcano, East Java, Indonesia was processed by the liquid mixture consisting of following twelve effective microorganisms (Rapha-el®; Lbiotech, Jeonnam, Korea), Lactobacillus parafarraginis, L. paracasei, L. harbinensis, L. buchneri, L. perolens, L. rhamnosus, L. vaccinostercus, Acetobacter lovaniensis, A. peroxydans, Pichia fermentans, Candida ethanolica, Saccharomycopsis schoenii isolated from fermentation process of oriental medicinal herbs including green tea, privet, and puer tea. The material was applied to in vitro antifungal activity test for Saprolegnia parasitica using agar dilution method. In addition, an acute toxicity test was performed on carp (Cyprinus carpio), eel (Anguilla japonica), and mud fish (Misgurnus mizolepis) for 96 hours. After three species of fish (n=15) were accustomed to experimental water environment for three days, the EM-PSS was added to each tank as final concentrations to be 0 to 500 ppm. The fish were taken into necropsy, and the histological sections of the gill, liver, and spleen were counter-stained with hematoxylin and eosin (H-E). And hence, no observed effect concentration (NOEC) of the solution was used for taking a medicinal bath for mudfish infected by Saprolegnia parasitica in practice. The result of in vitro antifungal activity test showed the growth inhibition of the fungus at 100 ppm, which and the lower concentrations occurred no fatal case in any fish species tested until the end of the examination. The 125 ppm of the solution, however, resulted in 13.3 %, 13.3 %, and 6.3 % of mortality in carp, eel, and mudfish, respectively. But both 250 and 500 ppm of the solution leaded lethality to all population of each fish species within 24 hours. Besides, H-E staining also showed no specific evidence for toxicity in fish at lesser than 100 ppm of EM-PSS. On the other hand, as a result of field application of the solution, no growth of fungal mycelium was found in fish bodies from gross observation 5 days post treatment. In conclusion, 100ppm of EM-PSS resulted in inhibition and treatment of Saprolegnia parasitica infection. In addition, the use of EM-PSS lower than 100 ppm is safe for fish. Therefore, EM-PSS could be used as aquatic fungicide, and also may be possible to be a potential eco-friendly disinfectant in aquaculture.

Keywords: antifungal activity, effective microorganism, toxicity, saprolegnia, processed sulfur solution

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Authors: Hyemi Kim, Jay Kim, Kyunghoon Han, Ra-Yeong Choi, In-Woo Kim, Hyung Joo Suh, Ki-Bae Hong, Sung Hee Han

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Chitosan is used in various industries such as food and medical care because it is known to have various functions such as anti-obesity, anti-inflammatory and anti-cancer benefits. Most of the commercial chitosan is extracted from crustaceans. As the harvest rate of snow crabs and red snow crabs decreases and safety issues arise due to environmental pollution, research is underway to extract chitosan from insects. In this study, we used Response Surface Methodology (RSM) to predict the optimal conditions to produce chitosan oligosaccharides from mealworms (MCOS), which can be absorbed through the intestine as low-molecular-weight chitosan. The experimentally confirmed optimal conditions for MCOS production using chitosanase were found to be a substrate concentration of 2.5%, enzyme addition of 30 mg/g and a reaction time of 6 hours. The chemical structure and physicochemical properties of the produced MCOS were measured using MALDI-TOF mass spectra and FTIR spectra. The MALDI-TOF mass spectra revealed peaks corresponding to the dimer (375.045), trimer (525.214), tetramer (693.243), pentamer (826.296), and hexamer (987.360). In the FTIR spectra, commercial chitosan oligosaccharides exhibited a weak peak pattern at 3500-2500 cm-1, unlike chitosan or chitosan oligosaccharides. There was a difference in the peak at 3200~3500 cm-1, where different vibrations corresponding to OH and amine groups overlapped. Chitosan, chitosan oligosaccharide, and commercial chitosan oligosaccharide showed peaks at 2849, 2884, and 2885 cm-1, respectively, attributed to the absorption of the C-H stretching vibration of methyl or methine. The amide I, amide II, and amide III bands of chitosan, chitosan oligosaccharide, and commercial chitosan oligosaccharide exhibited peaks at 1620/1620/1602, 1553/1555/1505, and 1310/1309/1317 cm-1, respectively. Furthermore, the solubility of MCOS was 45.15±3.43, water binding capacity (WBC) was 299.25±4.57, and fat binding capacity (FBC) was 325.61±2.28 and the solubility of commercial chitosan oligosaccharides was 49.04±9.52, WBC was 280.55±0.50, and FBC was 157.22±18.15. Thus, the characteristics of MCOS and commercial chitosan oligosaccharides are similar. The results of investigating the impact of chitosan oligosaccharide on the proliferation of probiotics revealed increased growth in L. casei, L. acidophilus, and Bif. Bifidum. Therefore, the major short-chain fatty acids produced by gut microorganisms, such as acetic acid, propionic acid, and butyric acid, increased within 24 hours of adding 1% (p<0.01) and 2% (p<0.001) MCOS. The impact of MCOS on the overall gut microbiota was assessed, revealing that the Chao1 index did not show significant differences, but the Simpson index decreased in a concentration-dependent manner, indicating a higher species diversity. The addition of MCOS resulted in changes in the overall microbial composition, with an increase in Firmicutes and Verrucomicrobia (p<0.05) compared to the control group, while Proteobacteria and Actinobacteria (p<0.05) decreased. At the genus level, changes in microbiota due to MCOS supplementation showed an increase in beneficial bacteria like lactobacillus, Romboutsia, Turicibacter, and Akkermansia (p<0.0001) while harmful bacteria like Enterococcus, Morganella, Proterus, and Bacteroides (p<0.0001) decreased. In this study, chitosan oligosaccharides were successfully produced under established conditions from mealworms, and these chitosan oligosaccharides are expected to have prebiotic effects, similar to those obtained from crabs.

Keywords: mealworms, chitosan, chitosan oligosaccharide, prebiotics

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Authors: Iñaki Iturria, Pasquale Russo, Montserrat Nacher-Vázquez, Giuseppe Spano, Paloma López, Miguel Angel Pardo

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Introduction: It is known that some Lactic Acid Bacteria (LAB) present an interesting probiotic effect. Probiotic bacteria stimulate host resistance to microbial pathogens and thereby aid in immune response, and modulate the host's immune responses to antigens with a potential to down-regulate hypersensitivity reactions. Therefore, probiotic therapy is valuable against intestinal infections and may be beneficial in the treatment of Inflammatory Bowel Disease (IBD). Several in vitro tests are available to evaluate the probiotic potential of a LAB strain. However, an in vivo model is required to understand the interaction between the host immune system and the bacteria. During the last few years, zebrafish (Danio rerio) has gained interest as a promising vertebrate model in this field. This organism has been extensively used to study the interaction between the host and the microbiota, as well as the host immune response under several microbial infections. In this work, we report on the use of the zebrafish model to investigate in vivo the colonizing ability and the immunomodulatory effect of probiotic LAB. Methods: Lactobacillus strains belonging to different LAB species were fluorescently tagged and used to colonize germ-free zebrafish larvae gastrointestinal tract (GIT). Some of the strains had a well-documented probiotic effect (L. acidophilus LA5); while others presented an exopolysaccharide (EPS) producing phenotype, thus allowing evaluating the influence of EPS in the colonization and immunomodulatory effect. Bacteria colonization was monitored for 72 h by direct observation in real time using fluorescent microscopy. CFU count per larva was also evaluated at different times. The immunomodulatory effect was assessed analysing the differential expression of several innate immune system genes (MyD88, NF-κB, Tlr4, Il1β and Il10) by qRT- PCR. The anti-inflammatory effect was evaluated using a chemical enterocolitis zebrafish model. The protective effect against a pathogen was also studied. To that end, a challenge test was developed using a fluorescently tagged pathogen (Vibrio anguillarum-GFP+). The progression of the infection was monitored up to 3 days using a fluorescent stereomicroscope. Mortality rates and CFU counts were also registered. Results and conclusions: Larvae exposed to EPS-producing bacteria showed a higher fluorescence and CFU count than those colonized with no-EPS phenotype LAB. In the same way, qRT-PCR results revealed an immunomodulatory effect on the host after the administration of the strains with probiotic activity. A downregulation of proinflammatory cytoquines as well as other cellular mediators of inflammation was observed. The anti-inflammatory effect was found to be particularly marked following exposure to LA% strain, as well as EPS producing strains. Furthermore, the challenge test revealed a protective effect of probiotic administration. As a matter of fact, larvae fed with probiotics showed a decrease in the mortality rate ranging from 20 to 35%. Discussion: In this work, we developed a promising model, based on the use of gnotobiotic zebrafish coupled with a bacterial fluorescent tagging in order to evaluate the probiotic potential of different LAB strains. We have successfully used this system to monitor in real time the colonization and persistence of exogenous LAB within the gut of zebrafish larvae, to evaluate their immunomodulatory effect and for in vivo competition assays. This approach could bring further insights into the complex microbial-host interactions at intestinal level.

Keywords: gnotobiotic, immune system, lactic acid bacteria, probiotics, zebrafish

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Authors: Veenu Bala, Yashpal S. Chhonker, Bhavana Kushwaha, Rabi S. Bhatta, Gopal Gupta, Vishnu L. Sharma

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According to UNAIDS 2013 estimate nearly 52% of all individuals living with HIV are now women of reproductive age (15–44 years). Seventy-five percent cases of HIV acquisition are through heterosexual contacts and sexually transmitted infections (STIs), attributable to unsafe sexual behaviour. Each year, an estimated 500 million people acquire atleast one of four STIs: chlamydia, gonorrhoea, syphilis and trichomoniasis. Trichomonas vaginalis (TV) is exclusively sexually transmitted in adults, accounting for 30% of STI cases and associated with pelvic inflammatory disease (PID), vaginitis and pregnancy complications in women. TV infection resulted in impaired vaginal milieu, eventually favoring HIV transmission. In the absence of an effective prophylactic HIV vaccine, prevention of new infections has become a priority. It was thought worthwhile to integrate HIV prevention and reproductive health services including unintended pregnancy protection for women as both are related with unprotected sex. Initially, nonoxynol-9 (N-9) had been proposed as a spermicidal agent with microbicidal activity but on the contrary it increased HIV susceptibility due to surfactant action. Thus, to accomplish an urgent need of novel woman controlled non-detergent microbicidal spermicides benzenepropanamine analogues have been synthesized. At first, five benzenepropanamine-dithiocarbamate hybrids have been synthesized and evaluated for their spermicidal, anti-Trichomonas and anti-fungal activities along with safety profiling to cervicovaginal cells. In order to further enhance the scope of above study benzenepropanamine was hybridized with thiourea as to introduce anti-HIV potential. The synthesized hybrid molecules were evaluated for their reverse transcriptase (RT) inhibition, spermicidal, anti-Trichomonas and antimicrobial activities as well as their safety against vaginal flora and cervical cells. simulated vaginal fluid (SVF) stability and pharmacokinetics of most potent compound versus N-9 was examined in female Newzealand (NZ) rabbits to observe its absorption into systemic circulation and subsequent exposure in blood plasma through vaginal wall. The study resulted in the most promising compound N-butyl-4-(3-oxo-3-phenylpropyl) piperazin-1-carbothioamide (29) exhibiting better activity profile than N-9 as it showed RT inhibition (72.30 %), anti-Trichomonas (MIC, 46.72 µM against MTZ susceptible and MIC, 187.68 µM against resistant strain), spermicidal (MEC, 0.01%) and antifungal activity (MIC, 3.12–50 µg/mL) against four fungal strains. The high safety against vaginal epithelium (HeLa cells) and compatibility with vaginal flora (lactobacillus), SVF stability and least vaginal absorption supported its suitability for topical vaginal application. Docking study was performed to gain an insight into the binding mode and interactions of the most promising compound, N-butyl-4-(3-oxo-3-phenylpropyl) piperazin-1-carbothioamide (29) with HIV-1 Reverse Transcriptase. The docking study has revealed that compound (29) interacted with HIV-1 RT similar to standard drug Nevirapine. It may be concluded that hybridization of benzenepropanamine and thiourea moiety resulted into novel lead with multiple activities including RT inhibition. A further lead optimization may result into effective vaginal microbicides having spermicidal, anti-Trichomonas, antifungal and anti-HIV potential altogether with enhanced safety to cervico-vaginal cells in comparison to Nonoxynol-9.

Keywords: microbicidal, nonoxynol-9, reverse transcriptase, spermicide

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Authors: Michelle Maurer, Antonia Last, Mark S. Gresnigt, Bernhard Hube, Alexander S. Mosig

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The intestinal epithelium forms an essential barrier to prevent translocation of microorganisms, toxins or other potentially harmful molecules into the bloodstream. In particular, dendritic cells of the intestinal epithelium orchestrate an adapted response of immune tolerance to commensals and immune defense against invading pathogens. Systemic inflammation is typically associated with a dysregulation of this adapted immune response and is accompanied by a disruption of the epithelial and endothelial gut barrier which enables dissemination of pathogens within the human body. To understand the pathophysiological mechanisms underlying the inflammation-associated gut barrier breakdown, it is crucial to elucidate the complex interplay of the host and the intestinal microbiome. A microfluidically perfused three-dimensional intestine-on-chip model was established to emulate these processes in the presence of immune cells, commensal bacteria, and facultative pathogens. Multi-organ tissue flow (MOTiF) biochips made from polystyrene were used for microfluidic perfusion of the intestinal tissue model. The biochips are composed of two chambers separated by a microporous membrane. Each chamber is connected to inlet and outlet channels allowing independent perfusion of the individual channels and application of microfluidic shear stress. Human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages and intestinal epithelial cells (Caco-2) were assembled on the biochip membrane. Following 7 – 14 days of growth in the presence of physiological flow conditions, the epithelium was colonized with the commensal bacterium Lactobacillus rhamnosus, while the endothelium was perfused with peripheral blood mononuclear cells (PBMCs). Additionally, L. rhamnosus was co-cultivated with the opportunistic fungal pathogen Candida albicans. Within one week of perfusion, the epithelial cells formed self-organized and well-polarized villus- and crypt-like structures that resemble essential morphological characteristics of the human intestine. Dendritic cells were differentiated in the epithelial tissue that specifically responds to bacterial lipopolysaccharide (LPS) challenge. LPS is well-tolerated at the luminal epithelial side of the intestinal model without signs of tissue damage or induction of an inflammatory response, even in the presence of circulating PBMC at the endothelial lining. In contrast, LPS stimulation at the endothelial side of the intestinal model triggered the release of pro-inflammatory cytokines such as TNF, IL-1β, IL-6, and IL-8 via activation of macrophages residing in the endothelium. Perfusion of the endothelium with PBMCs led to an enhanced cytokine release. L. rhamnosus colonization of the model was tolerated in the immune competent tissue model and was demonstrated to reduce damage induced by C. albicans infection. A microfluidic intestine-on-chip model was developed to mimic a systemic infection with a dysregulated immune response under physiological conditions. The model facilitates the colonization of commensal bacteria and co-cultivation with facultative pathogenic microorganisms. Both, commensal bacteria alone and facultative pathogens controlled by commensals, are tolerated by the host and contribute to cell signaling. The human intestine-on-chip model represents a promising tool to mimic microphysiological conditions of the human intestine and paves the way for more detailed in vitro studies of host-microbiota interactions under physiologically relevant conditions.

Keywords: host-microbiota interaction, immune tolerance, microfluidics, organ-on-chip

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