Arumugam Kamaladevi

Morin inhibits biofilm production and reduces the virulence of Listeria monocytogenes - An in vitro and in vivo approach.

The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100μg/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25μg/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM.

Bacillus amyloliquefaciens-secreted cyclic dipeptide – cyclo(L-leucyl-L-prolyl) inhibits biofilm and virulence production in methicillin-resistant Staphylococcus aureus

The current study explores the inhibitory efficacy of cyclo(L-leucyl-L-prolyl) (CLP), a cyclic dipeptide from Bacillus amyloliquefaciens on the biofilm and virulence production of methicillin-resistant Staphylococcus aureus (MRSA). The minimal inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) of CLP against three MRSA strains were found to be 256 and 512 μg mL−1 respectively. CLP at its sub-MICs (16, 32, 64 and 128 μg mL−1) exhibited a phenomenal dose-dependent antibiofilm activity against MRSA strains with maximum inhibitions of 85–87%. Confocal and scanning electron microscopic examinations validated the antibiofilm efficacy of CLP. In addition, CLP was proficient enough to greatly modify the surface hydrophobicity and significantly reduced the slime synthesis of MRSA. Appreciable differences noticed in the EPS constituents of CLP treated MRSA signified that the possible antibiofilm mechanism could proceed by impeding the synthesis of EPS and thereby CLP prevents biofilm assemblage and the associated virulence cascade. Interestingly, CLP displayed a prominent disruption (52–54%) on a 48 h preformed biofilm of MRSA. Data from in vivo assays using Caenorhabditis elegans unveiled the non-toxic and anti-infective efficacy of CLP. Down-regulation of all studied virulence genes affirmed the results of the phenotypic and in vivo assays. Thus, the present study exemplifies the use of CLP as a plausible alternative to conventional antibiotics in controlling biofilm associated infections of MRSA.

In Vitro and In Vivo Biofilm Characterization of Methicillin-Resistant Staphylococcus aureus from Patients Associated with Pharyngitis Infection

The present investigation was deliberately aimed at evaluating the biofilm-forming ability of 63 clinical MRSA isolates recovered from pharyngitis patients through different phenotypic assays. The molecular detection of adhesion (icaA/icaD/icaB/icaC), adhesins (fnbA/fnbB, clfA, and cna), staphylococcal accessory regulator (sarA), and α-toxin (hla) genes was done by employing polymerase chain reaction (PCR). Out of 63 isolates, 49 (77.8%) were found slime positive by the Congo red agar (CRA) method and 44 (69.8%) as biofilm positive by the quantitative microtitre plate assays. The results of MATH assay showed that most of the test pathogens are hydrophilic in nature. The molecular investigation of biofilm-associated genes revealed that 84.13% (n = 53) of isolates were found positive for icaADBC genes. The fnbA and fnbB genes were present in 49 (77.8%) and 51 (81%) MRSA isolates, respectively. In addition, 58.7% (n = 37), 73% (n = 46), and 69.8% (n = 44) of the isolates harboured the clfA, cna, and hla genes, respectively. Further, nearly 81% (n = 51) of the isolates were found positive for the gene sarA and all the ica negative isolates were also negative for the gene. Furthermore, the results of in vivo adherence assay unveiled the factual commonness in the in vitro adherence method.

Lactobacillus casei stimulates phase-II detoxification system and rescues malathion-induced physiological impairments in Caenorhabditis elegans

Malathion, an organophosphorus insecticide, is renowned for its inhibitory action on acetylcholinesterase (AChE) enzyme that eventually leads to widespread disturbance in the normal physiological and behavioral activities of any organism. Lactic acid bacteria (LAB) are still an underexploited and inexhaustible source of significant pharmaceutical thrust. In the present study, Caenorhabditis elegans was employed to identify and characterize the indigenous LAB isolated from different traditional food against malathion-induced toxicity. The results demonstrated that malathion at its LD50 concentration decreased various C. elegans physiological parameters such as survival, feeding, and locomotion. Among the screened isolates, L. casei exhibited an excellent protective efficacy against malathion-induced toxicity by increasing the level of AChE and thereby rescued all physiological parameters of C. elegans. In addition, short-term exposure and food choice assay divulged that L. casei could serve as a better food to protect C. elegans from noxious environment. The expression analysis unveiled that L. casei gavage upregulated the phase-II detoxification enzymes coding genes metallothioneins (mtl-1 and mtl-2) and glutathione-S-transferase (gst-8) and thereby eliminated malathion from the host system. Furthermore, the upregulation of ace-3 along with down-regulation of cyp35a in the nematodes supplemented with L. casei could be attributed to attenuate the malathion-induced physiological defects in C. elegans. Thus, the present study reports that an indigenous LAB-L. casei could serve as a promising protective agent against the harmful effects of pesticide.

Global Proteomics Revealed Klebsiella pneumoniae Induced Autophagy and Oxidative Stress in Caenorhabditis elegans by Inhibiting PI3K/AKT/mTOR Pathway during Infection

The enterobacterium, Klebsiella pneumoniae invades the intestinal epithelium of humans by interfering with multiple host cell response. To uncover a system-level overview of host response during infection, we analyzed the global dynamics of protein profiling in Caenorhabditis elegans using quantitative proteomics approach. Comparison of protein samples of nematodes exposed to K. pneumoniae for 12, 24, and 36 h by 2DE revealed several changes in host proteome. A total of 266 host-encoded proteins were identified by 2DE MALDI-MS/MS and LC-MS/MS and the interacting partners of the identified proteins were predicted by STRING 10.0 analysis. In order to understand the interacting partners of regulatory proteins with similar or close pI ranges, a liquid IEF was performed and the isolated fractions containing proteins were identified by LC-MS/MS. Functional bioinformatics analysis on identified proteins deciphered that they were mostly related to the metabolism, dauer formation, apoptosis, endocytosis, signal transduction, translation, developmental, and reproduction process. Gene enrichment analysis suggested that the metabolic process as the most overrepresented pathway regulated against K. pneumoniae infection. The dauer-like formation in infected C. elegans along with intestinal atrophy and ROS during the physiological analysis indicated that the regulation of metabolic pathway is probably through the involvement of mTOR. Immunoblot analysis supported the above notion that the K. pneumoniae infection induced protein mis-folding in host by involving PI3Kinase/AKT-1/mTOR mediated pathway. Furthermore, the susceptibility of pdi-2, akt-1, and mTOR C. elegans mutants confirmed the role and involvement of PI3K/AKT/mTOR pathway in mediating protein mis-folding which appear to be translating the vulnerability of host defense toward K. pneumoniae infection.

Lipopolysaccharide of Klebsiella pneumoniae attenuates immunity of Caenorhabditis elegans and evades by altering its supramolecular structure

Given the prominence of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative bacteria, investigations at the molecular level in in vivo conditions are in dire need to understand its role in provoking infection. Therefore, the current study was intentionally focused on LPS of Klebsiella pneumoniae to shed more light on its role in pathogenesis using an in vivo model system, Caenorhabditis elegans. In the killing assay, LPS showed a dose-dependent mortality in C. elegans. The gene expression analysis in wild-type (N2), sek-1, pmk-1 and tol-1 mutants unveiled that K. pneumoniae-LPS targeted the p38 MAPK pathway. In unison with this, the immunoblotting analysis further confirmed the requirement of a toll-dependent p38 MAPK pathway in nematode defense against K. pneumoniae-LPS. The MALDI-TOF analysis signified that K. pneumoniae escapes from the host defense by shuttering the rapid and prolonged immune activation by modifying its lipid A structure from the hexa-acylated (potent antagonist) to hepta-acylated form (weak antagonist). In addition, the increased number of acyl chain in fatty acids and the presence of palmitate, 4-aminoarabinose along with hepta-acylated lipid A (12 and 24 h) augmented the bacterial resistances against immune defense, which further supported the survival as well as multiplication of pathogen inside the host. Furthermore, the data on FT-IR and XRD unveiled that K. pneumoniae attaches to the host by establishing a strong hydrogen bond and thereby modifying its supramolecular structure of LPS from multilamellar (biologically non-active) to cubic/hexagonal (active), to evade the immune defense of the host and cause fatal infection.

Response of Caenorhabditis elegans during Klebsiella pneumoniae pathogenesis

Results K. pneumoniae and its LPS were lethal to C. elegans and required 48±5hours and 24±3hours for complete killing, respectively, with cessation of pharyngeal pumping and egg laying. Infection with K. pneumoniae increased the bacterial load in the intestine of host upon course of infection, which was measured as 1.5x10, 2.2x10, 3.6x10 and 3.7x10 in 4,6,12 and 24hours, respectively. This increased bacterial load subsequently disseminates oxidative stress markers in host. The level of ROS was measured to increase by 24.36597nM, 35.60517nM, 39.34052nM, and 28.24774 nM/mg of protein in 6, 12, 24, and 36 hours, respectively. Infection by K. pneumoniae also increased the protein carbonyls to 25.57nM, 36.14nM, 35.26nM and 38.84nM/mg of protein in 6, 12, 24, and 36 hours, respectively. K. pneumoniae and its LPS suppressed the expression of pmk-1, to l-1 and antimicrobial peptides (clec-60, clec-85, clec-87, lys-1, and lys-7) and thus succumbed the host by upregulated expression of virulent genes such as uge, rmpA and oxyR.