Mukesh Kumar Yadav

Eugenol: A Phyto-Compound Effective against Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Clinical Strain Biofilms

Background Inhibition and eradication of Staphylococcus aureus biofilms with conventional antibiotic is difficult, and the treatment is further complicated by the rise of antibiotic resistance among staphylococci. Consequently, there is a need for novel antimicrobials that can treat biofilm-related infections and decrease antibiotics burden. Natural compounds such as eugenol with anti-microbial properties are attractive agents that could reduce the use of conventional antibiotics. In this study we evaluated the effect of eugenol on MRSA and MSSA biofilms in vitro and bacterial colonization in vivo. Methods and Results Effect of eugenol on in vitro biofilm and in vivo colonization were studied using microtiter plate assay and otitis media-rat model respectively. The architecture of in vitro biofilms and in vivo colonization of bacteria was viewed with SEM. Real-time RT-PCR was used to study gene expression. Check board method was used to study the synergistic effects of eugenol and carvacrol on established biofilms. Eugenol significantly inhibited biofilms growth of MRSA and MSSA in vitro in a concentration-dependent manner. Eugenol at MIC or 2×MIC effectively eradicated the pre-established biofilms of MRSA and MSSA clinical strains. In vivo, sub-MIC of eugenol significantly decreased 88% S. aureus colonization in rat middle ear. Eugenol was observed to damage the cell-membrane and cause a leakage of the cell contents. At sub-inhibitory concentration, it decreases the expression of biofilm-and enterotoxin-related genes. Eugenol showed a synergistic effect with carvacrol on the eradication of pre-established biofilms. Conclusion/Major Finding This study demonstrated that eugenol exhibits notable activity against MRSA and MSSA clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may be used to control or eradicate S. aureus biofilm-related infections.

In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants.

Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC) and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA) ranging between 10–32 μg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 μg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 μg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM) and chitinase (chiC) were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L.) under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from within these plants and were shown to have antagonistic and plant growth promoting abilities. These results clearly suggest the possibility of using endophytic actinomycetes as bioinoculant for plant growth promotion, nutrient mobilization or as biocontrol agent against fungal phytopathogens for sustainable agriculture.

Role of Riboflavin in Induced Resistance against Fusarium Wilt and Charcoal Rot Diseases of Chickpea

Riboflavin caused induction of systemic resistance in chickpea against Fusarium wilt and charcoal rot diseases. The dose effect of 0.01 to 20 mM riboflavin showed that 1.0 mM concentration was sufficient for maximum induction of resistance; higher concentration did not increase the effect. At this concentration, riboflavin neither caused cell death of the host plant nor directly affected the pathogen`s growth. In time course observation, it was observed that riboflavin treated chickpea plants were inducing resistance 2 days after treatment and reached its maximum level from 5 to 7 days and then decreased. Riboflavin had no effect on salicylic acid (SA) levels in chickpea, however, riboflavin induced plants found accumulation of phenols and a greater activities of phenylalanine ammonia lyase (PAL) and pathogenesis related (PR) protein, peroxidase was observed in induced plant than the control. Riboflavin pre-treated plants challenged with the pathogens exhibited maximum activity of the peroxidases 4 days after treatment. Molecular weight of the purified peroxidase was 42 kDa. From these studies we demonstrated that riboflavin induced resistance is PRprotein mediated but is independent of salicylic acid.

Sinefungin, a Natural Nucleoside Analogue of S-Adenosylmethionine, Inhibits Streptococcus pneumoniae Biofilm Growth

Pneumococcal colonization and disease is often associated with biofilm formation, in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems, often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin, a nucleoside analogue of S-adenosylmethionine, on pneumococcal in vitro biofilm formation and in vivo colonization. Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS, pfs, and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo, decreases AI-2 production, and downregulates luxS, pfs, and speE gene expressions. Therefore, the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci.

Genetic diversity of Macrophomina phaseolina isolates from certain agro-climatic regions of India by using RAPD markers

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.

Daesiho-Tang Is an Effective Herbal Formulation in Attenuation of Obesity in Mice through Alteration of Gene Expression and Modulation of Intestinal Microbiota

Background Obesity has become a major global health challenge due to its increasing prevalence, and the associated health risk. It is the main cause of various metabolic diseases including diabetes, hypertension, cardiovascular disease, stroke and certain forms of cancer. Methods and Results In the present study we evaluated the anti-obesity property of Daesiho-tang (DSHT), an herbal medicine, using high fat diet (HFD)-induced obese mice as a model. Our results showed that DSHT ameliorated body weight gain, decreased total body fat, regulated expression of leptin and adiponectin genes of adipose tissue and exerted an anti-diabetic effect by attenuating fasting glucose level and serum insulin level in HFD-fed animals. In addition, DSHT-treatment significantly reduced total cholesterol (TC), triglycerides (TG) and increased high density lipoprotein-cholesterol (HDL), glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) levels in serum and reduced deposition of fat droplets in liver. DSHT treatment resulted in significantly increased relative abundance of bacteria including Bacteroidetes, Bacteroidetes/Firmicutes ratio, Akkermansia Bifidobacterium., Lactobacillus, and decreased the level of Firmicutes. Using RT2 profiler PCR array, 39 (46%) genes were found to be differentially expressed in HFD-fed mice compared to normal control. However, normal gene expressions were restored in 36 (92%) genes of HFD-fed mice, when co-exposed to DSHT. Conclusion/Major Findings The results of this study demonstrated that DSHT is an effective herbal formulation in attenuation of obesity in HFD-fed mice through alteration of gene expressions and modulation of intestinal microbiota.

In vitro Multi-Species Biofilms of Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa and Their Host Interaction during In vivo Colonization of an Otitis Media Rat Model

Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are known to cause biofilm-related infections. MRSA and PA have been frequently isolated from chronically infected wounds, cystic fibrosis, chronic suppurative otitis media (CSOM), and from indwelling medical devices, and these bacteria co-exist; however, their interaction with each-other or with the host is not well known. In this study, we investigated MRSA and PA multi-species biofilm communities in vitro and their interaction with the host during in vivo colonization using an OM rat-model. In-vitro biofilm formation and in-vivo colonization were studied using CV-microtiter plate assay and OM rat-model respectively. The biofilms were viewed under scanning electron microscope and bacteria were enumerated using cfu counts. The differential gene expressions of rat mucosa colonized with single or multi-species of MRSA or PA were studied using RNA-sequencing of total transcriptome. In multi-species in-vitro biofilms PA partially inhibited SA growth. However, no significant inhibition of MRSA was detected during in-vivo colonization of multi-species in rat bullae. A total of 1,797 genes were significantly (p < 0.05) differentially expressed in MRSA or PA or MRSA + PA colonized rat middle ear mucosa with respect to the control. The poly-microbial colonization of MRSA and PA induced the differential expression of a significant number of genes that are involved in immune response, inflammation, signaling, development, and defense; these were not expressed with single species colonization by either MRSA or PA. Genes involved in defense, immune response, inflammatory response, and developmental process were exclusively up-regulated, and genes that are involved in nervous system signaling, development and transmission, regulation of cell growth and development, anatomical and system development, and cell differentiation were down-regulated after multi-species inoculation. These results indicate that poly-microbial colonization induces a host response that is different from that induced by single species infection.

The Small Molecule DAM Inhibitor, Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro.

Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered and attached to the bottom of the plate when cells were grown in the presence of pyrimidinedione. Scanning electron microscopy analysis demonstrated the absence of an extracellular polysaccharide matrix in pyrimidinedione-grown biofilms compared to control-biofilms. Pyrimidinedione also significantly inhibited MRSA, MSSA, and Staphylococcus epidermidis biofilm growth in vitro. Furthermore, pyrimidinedione does not exhibit eukaryotic cell toxicity. In a microarray analysis, 56 genes were significantly up-regulated and 204 genes were significantly down-regulated. Genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down regulated in pyrimidinedione-grown biofilms. This study demonstrated that the small molecule Dam inhibitor, pyrimidinedione, inhibits pneumococcal biofilm growth in vitro at concentrations that do not inhibit planktonic cell growth and down regulates important metabolic-, virulence-, competence-, and biofilm-related genes. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.

Antimicrobial and Antibiofilm Effects of Human Amniotic/Chorionic Membrane Extract on Streptococcus pneumoniae.

Background: Streptococcus pneumoniae colonize the human nasopharynx in the form of biofilms. The biofilms act as bacterial reservoirs and planktonic bacteria from these biofilms can migrate to other sterile anatomical sites to cause pneumonia, otitis media (OM), bacteremia and meningitis. Human amniotic membrane contains numerous growth factors and antimicrobial activity; however, these have not been studied in detail. In this study, we prepared amniotic membrane extract and chorionic membrane extract (AME/CME) and evaluated their antibacterial and antibiofilm activities against S. pneumoniae using an in vitro biofilm model and in vivo OM rat model. Materials and Methods: The AME/CME were prepared and protein was quantified using DCTM (detergent compatible) method. The minimum inhibitory concentrations were determined using broth dilution method, and the synergistic effect of AME/CME with Penicillin-streptomycin was detected checkerboard. The in vitro biofilm and in vivo colonization of S. pneumoniae were studied using microtiter plate assay and OM rat model, respectively. The AME/CME-treated biofilms were examined using scanning electron microscope and confocal microscopy. To examine the constituents of AME/CME, we determined the proteins and peptides of AME/CME using tandem mass tag-based quantitative mass spectrometry. Results: AME/CME treatment significantly (p < 0.05) inhibited S. pneumoniae growth in planktonic form and in biofilms. Combined application of AME/CME and Penicillin-streptomycin solution had a synergistic effect against S. pneumoniae. Biofilms grown with AME/CME were thin, scattered, and unorganized. AME/CME effectively eradicated pre-established pneumococci biofilms and has a bactericidal effect. AME treatment significantly (p < 0.05) reduced bacterial colonization in the rat middle ear. The proteomics analysis revealed that the AME/CME contains hydrolase, ribonuclease, protease, and other antimicrobial proteins and peptides. Conclusion: AME/CME inhibits S. pneumoniae growth in the planktonic and biofilm states via its antimicrobial proteins and peptides. AME/CME are non-cytotoxic, natural human product; therefore, they may be used alone or with antibiotics to treat S. pneumoniae infections.

CST, an Herbal Formula, Exerts Anti-Obesity Effects through Brain-Gut-Adipose Tissue Axis Modulation in High-Fat Diet Fed Mice

The brain, gut, and adipose tissue interact to control metabolic pathways, and impairment in the brain-gut-adipose axis can lead to metabolic disorders, including obesity. Chowiseungcheng-tang (CST), a herbal formulation, is frequently used to treat metabolic disorders. Here, we investigated the anti-obesity effect of CST and its link with brain-gut-adipose axis using C57BL/6J mice as a model. The animals were provided with a normal research diet (NRD) or high-fat diet (HFD) in absence or presence of CST or orlistat (ORL) for 12 weeks. CST had a significant anti-obesity effect on a number of vital metabolic and obesity-related parameters in HFD-fed mice. CST significantly decreased the expression levels of genes encoding obesity-promoting neuropeptides (agouti-related peptide, neuropeptide Y), and increased the mRNA levels of obesity-suppressing neuropeptides (proopiomelanocortin, cocaine-and amphetamine-regulated transcript) in the hypothalamus. CST also effectively decreased the expression level of gene encoding obesity-promoting adipokine (retinol-binding protein-4) and increased the mRNA level of obesity-suppressing adipokine (adiponectin) in visceral adipose tissue (VAT). Additionally, CST altered the gut microbial composition in HFD groups, a phenomenon strongly associated with key metabolic parameters, neuropeptides, and adipokines. Our findings reveal that the anti-obesity impact of CST is mediated through modulation of metabolism-related neuropeptides, adipokines, and gut microbial composition.