Alka Narula

Attenuation of adhesion, quorum sensing and biofilm mediated virulence of carbapenem resistant Escherichia coli by selected natural plant products.

The multi-drug resistance offered by Carbapenem Resistant Escherichia coli (Family: Enterobacteriaceae; Class: Gammaproteobacteria) against third line antibiotics can be attributed towards its ability to develop biofilm. Such process involves adhesion and quorum-sensing induced colonization leading to biomass development. The present study explored the anti-adhesion, anti-quorum sensing and anti-biofilm potential of 05 pre-standardized potent herbals. Berberis aristata (PTRC-2111-A) exhibited maximum potential in all these activities i.e. 91.3% ± 0.05% (Anti-adhesion), 96.06% ± 0.05% (Anti-Quorum sensing) and 51.3% ± 0.07% (Anti-Biofilm formation) respectively. Camellia sinensis (PTRC-31911-A) showed both anti-adhesion (84.1% ± 0.03%) and anti-quorum sensing (90.0%) potential while Holarrhena antidysenterica (PTRC-8111-A) showed only anti-quorum sensing potential as compared to standards/antibiotics. These findings were in line with the molecular docking analysis of phytoligands against Lux S and Pilin receptors. Furthermore, the pairwise correlation analysis of the tested activities with qualitative, quantitative and bioactivity functional descriptors revealed that an increased content of alkaloid, moderate content of flavonoids and decreased content of tannins supported all the three activities. In addition, nitric oxide and superoxide scavenging activity were found to be correlated with anti-quorum sensing activity. The findings indicated clearly that B. aristata (Family: Berberidaceae) and C. sinensis (Family: Theaceae) were potent herbal leads with significant therapeutic potential which further needs to be explored at pre-clinical level in the future.

Camellia sinensis Ameliorates the Efficacy of Last Line Antibiotics Against Carbapenem Resistant Escherichia coli.

Aquo‐ethanolic extract of Camellia sinensis (PTRC‐31911‐A), standardized using Fourier transform infrared analysis, was found to have seven common functional groups in comparison with pre‐identified marker compound ‘quercetin’. Phyto‐chemical quantitation analysis revealed the presence of 10.65 µg/mg of flavonoids. The bioactivity fingerprint profile of PTRC‐31911‐A includes IC50 (Hydroxyl radical site specific scavenging) = 11.36 ± 0.5 µg/mL, IC80 (Hydroxyl radical non‐site specific scavenging) = 26.44 ± 0.5 µg/mL and IC50 (Superoxide ion scavenging) = 10.141 ± 0.5 µg/mL. The drug combination analysis of PTRC‐31911‐A with five third‐line antibiotics was carried out against carbapenem‐resistant Escherichia coli. The analysis of combination of PTRC‐31911‐A (6.25–1000 µg/mL) and antibiotics (6.25–1000 µg/mL) revealed synergistic behaviour (fractional inhibitory concentration indices < 1) with tigecycline, ertapenem, meropenem, colistin and augmentin. The lead combination of PTRC‐31911‐A + ertapenem or meropenem showed maximum augmentative potential at 50 and 100 µg/mL, respectively, with nearly five‐fold decrease in minimum inhibitory concentrations as compared with respective antibiotics alone. The synergistic effects implied that the antibacterial combinations of PTRC‐31911‐A and ertapenem, meropenem, colistin, tigecycline or augmentin would be more effective than a single monotherapy with either of the antibacterial agent. Copyright

Magnaporthe oryzae aminosugar metabolism is essential for successful host colonization.

Pathogens encounter and metabolize a range of host-derived metabolites while proliferating inside the host. Our understanding of these metabolites and their metabolic processes has remained largely incomplete. We investigated the role of the Magnaporthe oryzae N-acetylglucosamine (GlcNAc) catabolic pathway during rice infection. The catabolic pathway is composed of a GlcNAc transporter (MoNgt1), hexokinase(s), a GlcNAc-6-phosphate deacetylase (MoDac) and a GlcN-6-phosphate deaminase (MoDeam). A detailed characterization of the Δmongt1, Δmodac and Δmodeam null mutants revealed that a defect in GlcNAc catabolism impairs the pathogenicity of M. oryzae. These mutants showed severely reduced virulence in susceptible rice cultivar due to their inability to neutralize host-derived reactive oxygen species and their failure to develop invasive hyphal growth within the host tissue. Interestingly, during oxidative stress, M. oryzae proliferated efficiently in GlcNAc-containing media compared with other sugars, and the expression of fungal antioxidant genes was upregulated following GlcNAc treatment. However, GlcNAc inhibited the growth of the Δmodac and Δmodeam mutants, and this growth inhibition was enhanced during oxidative stress. These results suggest that GlcNAc helps fungus to overcome oxidative stress inside its host, perhaps by activating an antioxidant defence. In the absence of a functional catabolic pathway, GlcNAc becomes toxic to the cells.

Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants

Medicinal plants are one of the most important groups of plant genetic resources. Their use in biotechnology has assumed considerable significance because of overexploitation of these plants to meet the increasing demand. As cells cultured in vitro are prone to spontaneous changes, continuous culture of plant cells is often undesirable. Cryopreservation is a safe and cost-effective technique for preservation of germplasm and management of in vitro produced materials for biotechnological applications. The present article is a brief account of cryopreservation techniques and their application for medicinal plant conservation.

Augmenting the potency of third-line antibiotics with Berberis aristata: In vitro synergistic activity against carbapenem-resistant Escherichia coli

The aim of this study was to analyse the in vitro synergistic antibacterial potential of an aquoethanolic extract of the stem bark of Berberis aristata (PTRC-2111-A) with third-line antibiotics against carbapenem-resistant Escherichia coli. PTRC-2111-A was prepared and was characterised using phytochemical- and bioactivity-based fingerprinting. Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-mass spectrometry (LC-MS) analyses were performed, and superoxide and hydroxyl scavenging activities were assessed in conjunction with in vitro antimicrobial efficacy testing against the test micro-organism. Analysis of drug combinations of PTRC-2111-A and third-line antibiotics was performed using CompuSyn software. PTRC-2111-A from B. aristata was found to have seven common functional groups in comparison with the pre-identified marker compound quercetin, and phytochemical quantitation analysis revealed the presence of 25.44% alkaloids. Moreover, PTRC-2111-A was found to contain isoquinoline alkaloids, namely berbamine, berberine, reticuline, jatrorrhizine, palmatine and piperazine, as elucidated in the LC-MS analysis. Analysis of combinations of PTRC -2111-A and antibiotics revealed synergistic behaviour [fractional inhibitory concentration index (FICI)<1] with colistin, tigecycline and amoxicillin/clavulanate potassium (Augmentin(®)), whereas antagonism (FICI>1) was seen with ertapenem and meropenem.

Protective effect of Berberis aristata against peritonitis induced by carbapenem-resistant Escherichia coli in a mammalian model

OBJECTIVES Berberis aristata is known to contain a variety of phenolic compounds contributing to its holistic capability of mitigating bacterial multidrug resistance. METHODS B. aristata stem bark extract was prepared and was characterised using liquid chromatography-mass spectrometry (LC-MS). The antimicrobial efficacy of the extract against carbapenem-resistant Escherichia coli was assessed in vivo in an animal model using Sprague Dawley rats. Microbial counts in blood and urine, physical health status, haematological and biochemical analysis of blood, and histopathology of the kidney were assessed as the study endpoints. RESULTS An aquo-alcoholic extract of B. aristata (PTRC-2111-A) was found to effectively manage peritonitis induced by carbapenem-resistant E. coli in a rat model at a single post-exposure prophylactic dose of 0.5mg/kg body weight (BW). The extract was also found to show a no observed adverse effect level (NOAEL) up to a dose of 2000mg/kg BW. Physical, immunological, haematological, biochemical and histopathological aberrations were found to be restored to normal in the herbal-treated group at a dose of 0.5mg/kg BW. CONCLUSIONS The antimicrobial and hepatorenal protective ability of PTRC-2111-A could be attributed to the presence of isoquinoline alkaloids.

Effect of aquo-alchoholic extract of Glycyrrhiza glabra against Pseudomonas aeruginosa in Mice Lung Infection Model

The prevalence of lung infection caused by Pseudomonas aeruginosa strains that are classified as multi-drug resistant has increased considerably and is mainly attributed to relative insufficiency of potent chemotherapeutic modalities. The present study was conducted to evaluate the antimicrobial activity of aquo-alcoholic extract of Glycyrrhiza glabra against the P. aeruginosa causing lung infection in Swiss albino mice. The study involves evaluation of lethal dose of P. aeruginosa in Swiss albino mice and analysis of disease manifestation that includes bacteremia, hypothermia, reduction in body weight and other parameters for 48h of infection. Physical manifestations of infected mice showed a significant decline in body temperature that is 29±0.57°C (at 48th h) from 38.81±0.33°C (0h) and 30% weight loss was observed at the end of the study. Further the efficacy of G. glabra extract against lung infection induced with the calculated lethal dose was evaluated by employing bacteremia, histopathology and radiological analysis. Bacterial burden showed that 2.30±0.02 Log10CFU/mL at day 7, a significant decline in the bacterial load as compared to day 1 when the bacterial burden was found to be 3.32±0.1 Log10CFU/mL. Histopathological results showed more diffuse and patchy accumulation of inflammatory cells within the alveolar space also the infiltrates were noted in all the lung section of infected mice. In treated animal group improved lung histology was seen with the exudates were less seen in D1 dose (20mg/kg) and disappeared in D2 dose (80mg/kg). The study clearly declares that the G. glabra extract is effective against lung infection caused by P. aeruginosa at dose of 80mg/kg. The LCMS results revealed that the extract contains Glycyrrhizin, Stigmasterol and Ergosterol, Licochalcone and Glabridin. The current study expected to further exploit the biomedical properties of this extract in the preparation of a potent regimen against such threatening pathogen.

Assessment of aquo-ethanolic extract of Camellia sinensis against Carbapenem Resistant Escherichia coli: In Vivo Trials in a Murine Model.

The prevalence of Carbapenem Resistant Escherichia coli (CRE) has increased considerably during the last decade, which can be ascribed to relative scarcity of effective non toxic antimicrobial agents. The present study was conducted to evaluate the antimicrobial activity of aquo-ethanolic (1:1) extract of leaves of Camellia sinensis (PTRC-31911-A) against Carbapenem Resistant Escherichia coli at preclinical level using peritonitis infection model in Sprague Dawley rats. Efficacy analysis of PTRC-31911-A involved enumeration of CRE colonies in blood and urine samples of test animals for a period of 5 days from infection. A reduction in microbial count of biological fluids was considered as the primary endpoint of the selected murine model. Physical, biochemical, hematological and histological indices of toxicity were employed as secondary relative indicators of the induced disease. Physical manifestations of infected rats included significantly high body temperature (TempInfected=103.18°F, ∼5% increase) and noteworthy reduction in weight (WeightInfected=126.83g, ∼15% decrease) as compared to control. Significant (P<0.05) increase in total white blood cells, eosinophil and monocyte counts as well as a significant decrease (P<0.05) in erythrocytes count, hematocrit volume, red blood cell distribution width and hemoglobin concentration were observed in the infected group as compared to the control group. Furthermore, noteworthy increase in liver and kidney function test parameters were observed in case of infected groups. All the hematological and biochemical parameters were found to be within optimum range in case of treatment group, indicating restoration of homeostasis. Histopathological studies also presented symptoms of hemorrhage and glomerular damage with structural distortion in glomerular capillary loops of infected groups, which were later recovered in treated groups, indicating the nephro-protective potential of PTRC-31911-A. The study clearly points out that Camellia sinensis extract (PTRC-31911-A; single dose of 5mg/Kg bwt; oral,+24h) is highly effective against Carbapenem Resistant Escherichia coli owing mainly to the presence of flavonoids and polyphenolic compounds, identified by LCMS. Ongoing studies are expected to further unravel the mechanism of action and bioactivity determinants of this broad spectrum plant extract.

A comprehensive analysis of Candida albicans phosphoproteome reveals dynamic changes in phosphoprotein abundance during hyphal morphogenesis

The morphological plasticity of Candida albicans is a virulence determinant as the hyphal form has significant roles in the infection process. Recently, phosphoregulation of proteins through phosphorylation and dephosphorylation events has gained importance in studying the regulation of pathogenicity at the molecular level. To understand the importance of phosphorylation in hyphal morphogenesis, global analysis of the phosphoproteome was performed after hyphal induction with elevated temperature, serum, and N-acetyl-glucosamine (GlcNAc) treatments. The study identified 60, 20, and 53 phosphoproteins unique to elevated temperature-, serum-, and GlcNAc-treated conditions, respectively. Distribution of unique phosphorylation sites sorted by the modified amino acids revealed that predominant phosphorylation occurs in serine, followed by threonine and tyrosine residues in all the datasets. However, the frequency distribution of phosphorylation sites in the proteins varied with treatment conditions. Further, interaction network-based functional annotation of protein kinases of C. albicans as well as identified phosphoproteins was performed, which demonstrated the interaction of kinases with phosphoproteins during filamentous growth. Altogether, the present findings will serve as a base for further functional studies in the aspects of protein kinase-target protein interaction in effectuating phosphorylation of target proteins, and delineating the downstream signaling networks linked to virulence characteristics of C. albicans.

In vitro bactericidal activity of Berberis aristata extract against clinical isolates of carbapenem-resistant Escherichia coli.

Abstract Background: Berberis aristata is known to contain a variety of phenolic compounds, flavonoids such as quercetin attributing towards its holistic capability of mitigating multidrug resistance. Methods: B. aristata stem bark extract was prepared and characterized using phytochemical and bioactivity-based fingerprinting. Anti-oxidant and anti-lipid peroxidation profiling was also done in conjunction with in vitro anti-microbial efficacy testing against the test microorganism i. e., New Delhi Metallo-β-lactamase-1 (NDM-1) Escherichia coli. Results: Aquo-alcoholic (1:1) extract of B. aristata (PTRC-2111-A), containing 3.0±0.02 µg of QUERCETIN/mg of dried extract, exhibited [flavonoid/polyphenol: F/P (quercetin %) ~ 0.16(0.06 %)]. The bioactivity fingerprint profile of PTRC-2111-A included IC50 ratio [DPPH/NOS]=0.064 as functional standardized value having IC50 (DPPH Scavenging)=16±0.5 µg/mL and IC50 (Nitric Oxide Scavenging)=250±0.5 µg/mL respectively. The reducing ability and anti-lipid peroxidation equivalent (extract: standard) of PTRC-2111-A with respect to standard was estimated to be 3.44 (ascorbic acid) and 0.78 (quercetin) respectively. In vitro anti-microbial activity evaluated against sts-09 multidrug-resistant strain of carbapenem-resistant E. coli was found to be 25 µg/mL. Conclusions: B. aristata was found to contain a number of phytoconstituents, which acts in a synergistic manner to provide significant bactericidal potential against carbapenem-resistant E. coli.