Chandradipa Ghosh

The Sodium-Driven Flagellar Motor Controls Exopolysaccharide Expression in Vibrio cholerae

Vibrio cholerae causes the life-threatening diarrheal disease cholera. This organism persists in aquatic environments in areas of endemicity, and it is believed that the ability of the bacteria to form biofilms in the environment contributes to their persistence. Expression of an exopolysaccharide (EPS), encoded by two vps gene clusters, is essential for biofilm formation and causes a rugose colonial phenotype. We previously reported that the lack of a flagellum induces V. cholerae EPS expression. To uncover the signaling pathway that links the lack of a flagellum to EPS expression, we introduced into a rugose flaA strain second-site mutations that would cause reversion back to the smooth phenotype. Interestingly, mutation of the genes encoding the sodium-driven motor (mot) in a nonflagellated strain reduces EPS expression, biofilm formation, and vps gene transcription, as does the addition of phenamil, which specifically inhibits the sodium-driven motor. Mutation of vpsR, which encodes a response regulator, also reduces EPS expression, biofilm formation, and vps gene transcription in nonflagellated cells. Complementation of a vpsR strain with a constitutive vpsR allele likely to mimic the phosphorylated state (D59E) restores EPS expression and biofilm formation, while complementation with an allele predicted to remain unphosphorylated (D59A) does not. Our results demonstrate the involvement of the sodium-driven motor and suggest the involvement of phospho-VpsR in the signaling cascade that induces EPS expression. A nonflagellated strain expressing EPS is defective for intestinal colonization in the suckling mouse model of cholera and expresses reduced amounts of cholera toxin and toxin-coregulated pili in vitro. Wild-type levels of virulence factor expression and colonization could be restored by a second mutation within the vps gene cluster that eliminated EPS biosynthesis. These results demonstrate a complex relationship between the flagellum-dependent EPS signaling cascade and virulence.

Coronary Artery Disease Risk Factors & Their Association with Physical Activity in Older Athletes

Objective To evaluate the effects of present-day physical activity on selected coronary artery disease (CAD) risk factors of older former athletes and to compare these selected risk factors with age-matched older non-athletes. Methods The selected CAD risk factors were compared among the active older athletes (n = 52; 45.9 ± 4.75 years), sedentary older athletes (n = 54; 47.2 ± 4.67 years), and sedentary older non-athletes (n = 56; 46.0 ± 5.26 years) of Calcutta and surroundings. CAD risk factors including anthropometric obesity parameters, blood lipids and blood pressure were measured. The present-day total physical activity of each individual was assessed by a questionnaire and estimated through the sum of energy expended during habitual, professional, recreational and conditioning physical activities, and sleeping time. The total physical activity was expressed as energy expended in mega joules per kilogram of body weight per week (MJ.kg−1.week−1). Maximal oxygen consumption (VO2 max) was assessed by maximal treadmill test. Body score distributions in each group were also calculated. Results The selected CAD risk factors differed significantly among the groups. The sedentary older athletes had significantly higher mean values in weight, BMI, body fat percentage, total cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides and ratio of total cholesterol to high-density lipoprotein cholesterol (total C/HDLC) than that of active older athletes and sedentary older non-athletes. A reverse trend was observed in the case of HDL cholesterol. On the other hand, the presently active older athletes had significantly favourable levels of most of the selected CAD risk factors than the sedentary older athletes and non-athletes. The present-day total physical activity had significant negative association with total cholesterol, ratio of total cholesterol to HDL cholesterol, LDL cholesterol, triglycerides, resting systolic blood pressure when controlling for the effects of age, body mass index and body fat percentages. So, it is confirmed from this analysis that the subjects in the present study who had a greater present-day total physical activity had favourable CAD risk factors. Conclusions The results of this study indicate that the risk for CAD or levels of selected CAD risk factors of former athletes after retirement from active sports were more related to the present-day physical activity i.e., higher total physical activity lowers the risk of coronary artery disease.

Biochemical and Molecular Analysis of Staphylococcus aureus Clinical Isolates from Hospitalized Patients

Staphylococcus aureus is opportunistic human as well as animal pathogen that causes a variety of diseases. A total of 100 Staphylococcus aureus isolates were obtained from clinical samples derived from hospitalized patients. The presumptive Staphylococcus aureus clinical isolates were identified phenotypically by different biochemical tests. Molecular identification was done by PCR using species specific 16S rRNA primer pairs and finally 100 isolates were found to be positive as Staphylococcus aureus. Screened isolates were further analyzed by several microbiological diagnostics tests including gelatin hydrolysis, protease, and lipase tests. It was found that 78%, 81%, and 51% isolates were positive for gelatin hydrolysis, protease, and lipase activities, respectively. Antibiogram analysis of isolated Staphylococcus aureus strains with respect to different antimicrobial agents revealed resistance pattern ranging from 57 to 96%. Our study also shows 70% strains to be MRSA, 54.3% as VRSA, and 54.3% as both MRSA and VRSA. All the identified isolates were subjected to detection of mecA, nuc, and hlb genes and 70%, 84%, and 40% were found to harbour mecA, nuc, and hlb genes, respectively. The current investigation is highly important and informative for the high level multidrug resistant Staphylococcus aureus infections inclusive also of methicillin and vancomycin.

Effects of secondary carbon supplement on biofilm-mediated biodegradation of naphthalene by mutated naphthalene 1, 2-dioxygenase encoded by Pseudomonas putida strain KD9

Polycyclic aromatic hydrocarbons (PAHs) belong to a diverse group of environmental pollutants distributed ubiquitously in the environment. The carcinogenic properties of PAHs are the main causes of harm to human health. The green technology, biodegradation have become convenient options to address the environmental pollution. In this study, we analyzed the biodegradation potential of naphthalene with secondary carbon supplements (SCSs) in carbon deficient media (CSM) by Pseudomonas putida strain KD9 isolated from oil refinerary waste. The rigid-flexible molecular docking method revealed that the mutated naphthalene 1,2-dioxygenase had lower affinity for naphthalene than that found in wild type strain. Moreover, analytical methods (HPLC, qRT-PCR) and soft agar chemotaxis suggest sucrose (0.5 wt%) to be the best chemo-attractant and it unequivocally caused enhanced biodegradation of naphthalene (500 mg L-1) in both biofilm-mediated and shake-flask biodegradation methods. In addition, the morphological analysis detected from microscopy clearly showed KD9 to change its size and shape (rod to pointed) during biodegradation of naphthalene in CSM as sole source of carbon and energy. The forward versus side light scatter plot of the singlet cells obtained from flow cytometry suggests smaller cell size in CSM and lower florescence intensity of the total DNA content of cells. This study concludes that sucrose may be used as potential bio-stimulation agent.

Quorum Sensing Autoinducer(s) and Flagellum Independently Mediate EPS Signaling in Vibrio cholerae Through LuxO-Independent Mechanism

Vibrio cholerae, the Gram-negative bacterium causing lethal diarrheal disease cholera, forms biofilm on solid surfaces to gain adaptive advantage for successful survival in aquatic reservoirs. Expression of exopolysaccharide (EPS), an extracellular matrix material, has been found critical for biofilm-based environmental persistence. In a subset of epidemic-causing V. cholerae, absence of flagellum but not motility was identified to induce elevated exopolysaccharide expression. Identification of the role played by quorum sensing autoinducer molecules, i.e., cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2) as well as central regulator LuxO on EPS expression in the subset was explored. Deletion mutations were introduced in vital genes responsible for synthesizing CAI-1 (cqsA), AI-2 (luxS), flagellum (flaA), LuxO (luxO), flagellar motor (motX), and VpsR (vpsR) in the model strain MO10. Subsequent phenotypic alterations in terms of colony morphology, EPS expression, biofilm formation, and transcription level of relevant genes were analyzed. Autoinducer cross-feeding experiment confirmed the role of autoinducers in EPS signaling. Results reveal that autoinducers and flagellum are the two major EPS signaling units in this subset where one unit becomes predominant for EPS production in absence of the other. Moreover, either unit exerts negative influence on EPS induction by the other. Both the EPS signaling cascades are independent of LuxO contribution and essentially involve sodium-driven flagellar motor and VpsR. A cell density and flagellum-mediated, but LuxO-independent, EPS signaling mechanism is considered to be functional in these organisms that confers their survival fitness.

Prevalence of Panton-Valentine Leukocidin Gene among Community Acquired Staphylococcus aureus: A Real-Time PCR Study

Panton-Valentine leukocidin (luk-pv) is a cytotoxin that causes leukocyte destruction and tissue necrosis. The aim of this study was to determine the prevalence of the pv1, mecA, and nuc genes in Staphylococcus aureus isolates obtained from anterior nares and superficial infection sites of skin in a slum population of West Bengal, India. Expression level of pv1 gene was also analysed. Twenty-two S. aureus strains were isolated, and phenotype and genotype specific examinations for S. aureus isolates were carried out. Molecular identification was done by PCR using species-specific 16S rRNA primer pairs and finally 22 isolates were found to be positive as S. aureus. The antibiotic responsiveness of all these isolates and the minimum inhibitory concentration (MIC) of MRSA isolates were determined using the broth dilution method with vancomycin. Antibiogram analysis of isolated S. aureus strains with respect to different antimicrobial agents revealed antibiotic resistance ranging from 27 to 91%. The results of MIC for vancomycin showed 95% of strains to be VSSA and 5% to be VISA. 68% isolates were resistant to methicillin. All the isolates were subjected to detection of pv1, mecA, and nuc genes, and 9%, 68%, and 27% were found to harbour pvl, mecA, and nuc genes, respectively. All the MRSA strains produced high to moderate levels of biofilm. pvl gene expression was carried out in vitro by Real-Time PCR. The low ∆Ct value (0.493) was indicative of high expression of pvl in one S. aureus strain. Thus, detection of pvl gene in community acquired S. aureus indicates the emergence of pathogenic S. aureus in community setup in the studied region. The existing exploration is extremely imperative and informative for the high level multi-drug resistant S. aureus infections inclusive of MRSA.

Paradoxical Bactericidal Effects of Hydrophobic Lung Surfactant Proteins and Their Peptide Mimics Using Liposome Molecular Trojan

Lung surfactant, besides alveolar stability, also provides defence against pathogens by surfactant proteins (SP), SP-A and SP-D. The hydrophobic proteins SP-B and SP-C enhance surface activity. An unusual and paradoxical effect of bovine LS and synthetic model LS with SP-B/-C was bactericidal to Staphylococcus aureus and Escherichia coli. Bacterial proliferation were investigated with bovine lung surfactant extract (BLES), dipalmitoylphosphatdylcholine, palmitooleylglycerol, in combination with SP-B/-C using standard microbiological colony forming unit (CFU) counts and structural imaging. BLES and other surfactant-SP-B/-C mixtures inhibit bacterial growth in the concentration range of 0 -7.5 mg/mL, at > 10 mg/mL paradoxical growth of both the bacterial species suggest antibiotic resistance. The lipid only LS have no effect on bacterial proliferation. Smaller peptide mimics of SP-B or SP-B1-25, were less efficient than SP-Cff. Ultra structural studies of the bacterial CFU using electron and atomic force microscopy suggest some membrane damage of S. aereus at inhibitory concentration of BLES, and some structural alteration of E. coli at dividing zones, suggesting utilization and incorporation of surfactant lipid species by both bacteria. The results depicted from in vitro studies are also in agreement with protein-protein interactions obtained from PatchDock, FireDock and ClasPro algorithm. The MD-simulation decipher a small range fluctuation of gyration radius of the LS proteins and their peptide mimics. The studies have alarming implications in the use of high dosages (100 mg/mL/kg body weight) of exogenous surfactant for treatment of respiratory distress syndrome, genetic knock-out abnormalities associated with these proteins, and the novel roles played by SP-B/C as bactericidal agents.

Effect of dietary deficiency of vitamin B6, vitamin B12 and folic acid on the development of coronary artery disease in adult Indian male patients

The present study was conducted to find out the correlation of dietary deficiency of vitamin B6, vitamin B12 and folic acid on development of coronary artery disease (CAD). This study was carried out on 172 male patients of age group between 45-65 years having both genetic and non-genetic background for CAD from Kolkata, India. Present study showed that BMI, SBP, DBP, LDL-c, triglyceride, total cholesterol, fasting and post prandial blood glucose level, homocysteine, CPK and CPK-MB enzyme levels were higher while HDL-c, LVEF and hemoglobin levels were lower than recommended normal values in both subject groups. Daily consumption of carbohydrate, vitamin B12, vitamin B6 and folic acid levels of both subject groups were found to be lower while consumption of fat was higher than recommended values. Present study showed that there was a significant correlation between intake of vitamin B6, vitamin B12 and folic acid and risk factors for CAD in both groups. All these findings indicate that folic acid, vitamin B6 and vitamin B12 deficiency may have influence in developing risk for CAD.