Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution.

Phage therapy has been at the centre of attraction for combating multi-drug resistant strains. However, less stability and rapid clearance of phage by mononuclear phagocytic system (MPS) restricts its use in humans. In the present study, aim was to develop a liposomal delivery system for bacteriophage that can assure efficient phage delivery and retention at the site of infection. Different ratios of cholesterol, lipids and surfactant along with different charge inducers were employed to prepare liposomes. Phage was then entrapped in the liposomes and characterized on the basis of morphology, size, entrapment efficiency and stability. Further, in vivo biodistribution of free phage and liposome entrapped phage was compared in different organs of mice. A cationic liposomal formulation showed maximum encapsulation efficiency of 92%. Transmission electron microscopy (TEM) confirmed the entrapment of phages in liposomes. Liposome preparation was found to be most stable at 4°C during storage. Liposome entrapped bacteriophage was retained for longer duration in different organs i.e. upto day 4 in blood, day 6 in liver, lungs and kidney, 14days in spleen of mice as compared to free phage that became undetectable by 36th h in blood as well as lungs and by 48th h in all other organs.

Ellagic acid derivatives from Terminalia chebula Retz. increase the susceptibility of Pseudomonas aeruginosa to stress by inhibiting polyphosphate kinase.

AIM: Polyphosphate kinase 1 (PPK1) plays an important role in virulence, antibiotic resistance and survival under stress conditions and, therefore, is an attractive therapeutic target to control infections caused by multiple drug resistant Pseudomonas aeruginosa. This study explores the PPK1 inhibiting activity of ellagic acid derivatives (EADs) from Terminalia chebula Retz. that could increase the susceptibility of Ps. aeruginosa to in vitro stress conditions. METHODS AND RESULTS: EADs reduced ppk1 gene expression by 93% (P < 0·05) and completely inhibited its activity (P < 0·01) at 0·5 mg ml(-1) . EADs-treated Ps. aeruginosa showed marked reduction in polyphosphate granules in cytosol. Expression of rpoS, the downstream master stress response regulator, was reduced by 94% (P < 0·05) and the sensitivity of Ps. aeruginosa increased many fold to desiccation, oxidative (H2 O2 ) and antibiotic (piperacillin) stresses. PPK-regulated swimming, swarming and twitching motilities and biofilm formation were also reduced significantly (P ≤ 0·05) in MPAO1 and the clinical strains of Ps. aeruginosa. CONCLUSION: EADs from T. chebula inhibited PPK1 expression and its activity and increased the sensitivity of Ps. aeruginosa to desiccation and oxidative stress while reducing tolerance to piperacillin. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of EADs as therapeutic agent against Ps. aeruginosa.

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection.

PURPOSE: Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. MATERIALS AND METHODS: An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. RESULTS: Both parent and mutant strains were able to reach the renal tissue. PAO 1 PAO1 was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. CONCLUSION: This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.

Enzymatic quorum quenching increases antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: There is increasing emergence of multidrug resistant Pseudomonas aeruginosa (MDRPA) strains and drug resistance is positively-correlated with biofilm-forming ability. Since about 10% of P. aeruginosa genome is controlled by quorum sensing (QS), alteration in its antibiotic susceptibility by targeting QS was the focus of the present study. MATERIALS AND METHODS: One day biofilms of PAO1 and three urinary tract infection MDRPA isolates (PA2, PA8 and PA18) were formed in 96-well microtiter plate. Biofilms were exposed to concentration gradient of ciprofloxacin and gentamicin to obtain Minimum Biofilm Eradication Concentration (MBEC) by direct enumeration method. Susceptibility of 24 h biofilms was evaluated by treatment with ciprofloxacin and gentamicin per se and in combination with lactonase. The effect was also examined on 72 h biofilms by Scanning Electron Microscopy. RESULTS: Lactonase treatment did not have any effect on growth of the selected strains but 73.42, 69.1, 77.34 and 72.5% reduction of biofilm was observed after lactonase (1 unit) treatment, respectively. Antibiotics in combination with lactonase (0.3 units) resulted in an increased susceptibility of the biofilm forms by>3.3, 4, 5 and 1.5 folds of MBEC, for ciprofloxacin and>6.67, 12.5, 6 and>2.5 folds, for gentamicin respectively, which could be due to the disruption of biofilm by lactonase treatment as shown by scanning electron microscopy. Also there was significant reduction (p<0.001) in virulence factor production by the strains. CONCLUSION: Lactonase treatment increased antibiotic susceptibility of the biofilms of MDRPA isolates underscoring the potential of quorum quenching in antimicrobial therapeutics.

Sea-cod oil supplementation alters the course of Streptococcus pneumoniae infection in BALB/c mice.

The existing reports on the role of ω-3 polyunsaturated fatty acids (PUFA) in infectious diseases are contradictory. The present study was conducted to evaluate the effect of sea-cod oil on the course of respiratory tract infection by Streptococcus pneumoniae in BALB/c mice. Animals were given enteral sea-cod oil for a period of 30 and 60 days and challenged intra-tracheally with S. pneumoniae D39 serotype 2. The survival of animals and various inflammatory parameters, i.e. myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide (NO) and leukotriene B(4) in the lung homogenates, were investigated. The inflammatory cytokines levels (IL-1ß, TNF-α and IL-10) were also determined. Continuous sea-cod oil supplementation for 60 days significantly improved survival among the animals. A significant reduction in the bacterial load in the lungs of sea-cod oil-fed animals compared to the controls was observed. As the disease progressed, the reduced bacterial colonisation correlated well with the histopathological observation. This was accompanied by a decrease in the production of inflammatory mediators and cytokines in the lung homogenates. However, not even a minor difference was seen in animals given sea-cod oil supplementation for 30 days duration; therefore, long-term treatment was required to attain an effect. Sea-cod oil supplementation modulated the host immune response and, thus, protected the host from ensuing inflammatory damage due to S. pneumoniae-mediated infection.

Isolation and characterization of Klebsiella pneumoniae specific bacteriophages from sewage samples.

Five bacteriophages (Kpn5, Kpn12, Kpn13, Kpn17 and Kpn22), each having specificity against Klebsiella pneumoniae strain B5055, were isolated from sewage samples and characterized in terms of growth characteristics, genetic material, morphology and structural proteins. Adsorption rate as well as single step growth curve experiments showed variation among phages. Restriction enzyme digestion of DNA confirmed the presence of double stranded DNA as well as the heterogeneous nature of genetic material. RAPD-PCR was performed to further distinguish these closely related phages. Their genome fingerprint confirmed their diversity. Transmission electron microscopy, on the other hand, showed their morphological similarity; they were assigned to family Podoviridae, order Caudovirales on the basis of their head and tail morphology. Structural proteins resolved on SDS-PAGE showed the presence of similar major outer membrane proteins. The bacteriophages, belonging to Podoviridae family with short stumpy tails, were found to be nontoxic to mice. They showed maximum count in various organs at 6 h post inoculation, which persisted till 36 h. These phages thus have the potential to be used for phage therapy.

Dual antiplatelet therapy for more than 12 months after percutaneous coronary intervention: insights from the Guthrie PCI Registry.

OBJECTIVE: To assess the impact of dual antiplatelet (DAP) therapy of >12 months on long-term death and myocardial infarction (MI) after percutaneous coronary intervention (PCI). DESIGN, SETTING AND PATIENTS: Prospective, single-centre, observational study of 1859 consecutive patients who underwent successful PCI of a native coronary artery and survived event-free for at least 12 months. MAIN OUTCOME MEASURES: Combined end point of death or non-fatal MI determined by survival analysis and propensity-adjusted multivariable Cox regression. Similar analyses were performed in the two stent subsets: bare metal stents (n = 835), drug-eluting stents (n = 1024); and three high-risk subsets: diabetic patients (n = 486), patients presenting with MI (n = 713), and those with ACC/AHA type C lesions (n = 717). RESULTS: Baseline characteristics were as follows: mean (SD) age 64 (12) years, male 69%, diabetic 26%, presentation with MI 38%, mean (SD) ejection fraction 49 (12)%, mean (SD) vessel diameter 3.1 (0.5) mm. Duration of DAP was 27 (11) months in "DAP >12 months" and 4.1 (4.1) months in "DAP < or =12 months" (p<0.001). At a median follow-up of 3.4 years after PCI, "DAP >12 months" vs "DAP < or =12 months" had similar incidence of death or MI (9.4% vs 10.3%, log-rank p = 0.83). After multivariable adjustment, DAP therapy >12 months was not associated with lower incidence of death or MI than DAP therapy < or =12 months (adjusted HR = 1.01; 95% CI 0.74 to 1.37, p = 0.95). Analysis of each of the five predefined subsets showed similar results. CONCLUSIONS: In patients who undergo successful native coronary PCI and survive event-free for at least 12 months, continuation of dual antiplatelet therapy beyond 12 months does not confer long-term protection from death or MI.

A combination of thalidomide and augmentin protects BALB/c mice suffering from Klebsiella pneumoniae B5055-induced sepsis.

Despite extensive research, the mortality associated with sepsis in hospitals remains very high. We have evaluated the protective immunomodulatory effect of thalidomide alone or with Augmentin in Klebsiella pneumoniae B5055-induced sepsis in BALB/c mice. The mouse model of sepsis was developed by placing K. pneumoniae B5055 entrapped in fibrin and thrombin clots in the peritoneal cavity of mice. The septic mice were treated with thalidomide alone (30 mg/kg/day/po), Augmentin alone (20 microg/ml/ip) and with their combination. the thalidomide-alone treated mice showed 75% survival whereas 60% of the Augmentin-alone treated group survived. Combination treatment provided 100% survival. Treatment with thalidomide alone significantly (p<0.05) decreased interleukin-1 alpha (IL-1alpha), nitric oxide (NO) and malondialdehyde (MDA) levels in the serum without significantly (p<0.05) decreasing the bacterial count in blood. Augmentin alone only decreased the bacterial load in blood significantly (p<0.05). However, a combination of thalidomide with Augmentin significantly (p<0.05) decreased both the bacterial count and inflammatory mediators.

Effect of clarithromycin on lung inflammation and alveolar macrophage function in Klebsiella penumoniae B5055-induced acute lung infection in BALB/c mice.

Acute lung injuries due to acute lung infections remain the major cause of mortality. Thus antibiotics with immunomodulatory and anti-inflammatory activities ,regardless of their antibacterial properties, will help to overcome acute lung infection-induced injuries. The macrolide antibiotics have been shown to possess these properties. Clarithromycin has been shown to possess anti-inflammatory properties in chronic inflammatory conditions. So we evaluated the anti-inflammatory effect of clarithromycin treatment in Klebsiella pneumoniae B5055-induced acute lung infection in mice. The clarithromycin treatment significantly (p<0.05) decreased the bacterial load in the lungs of K. pneumoniae B5055-infected mice and significantly (p<0.05) increased macrophage activity. The clarithromycin treatment also significantly ( p<0.05) decreased the neutrophil infiltration into the lungs and decreased myeloperoxidase (MPO) activity. Clarithromycin significantly (p<0.05) decreased malondialdehyde (MDA) and nitric oxide (NO) production and thus decreased acute lung injury occurring during acute lung infection.

Effect of pH on production of virulence factors by biofilm cells of Pseudomonas aeruginosa.

Effect of pH on production of extracellular virulence factors of Pseudomonas aeruginosa grown on catheter in biofilm was determined. Alginate and proteinase production was higher at pH 8; in contrast, siderophores (pyochelin and pyoverdin) were synthesized more intensively at pH 5.

Lipopolysaccharide-mediated protection against Klebsiella pneumoniae-induced lobar pneumonia: intranasal vs. intramuscular route of immunization.

Immunoprotective potential of delivered lipopolysaccharide (LPS) preparation from Klebsiella pneumoniae was determined in a murine model of lobar pneumonia. Protection was assessed with three doses of LPS (25, 50 and 100 microg; without any adjuvant) administered intranasally or intramuscularly. After evaluation of lung tissue (bacterial load and histopathology), no significant protection was observed at 25 microg with either application. A significant decrease in lung bacterial load coupled with fall in severity of lung lesions was observed with 50 microg (again both applications). At 100 microg dose, with intramuscular route, a further decrease in the lung bacterial load was shown compared to the 50 microg dose. In contrast, 100 microg LPS, when given intranasally, resulted in a higher bacterial colonization of the lung tissue and higher lung pathology; thus we recommend intramuscular instead of the intranasal route for developing protection against K. pneumoniae-mediated pneumonia with intact LPS-based vaccines.

Urovirulence of Pseudomonas aeruginosa: planktonic cells vs. biofilm cells.

Planktonic and biofilm cells of a clinical urinary isolate of P. aeruginosa were compared in vitro for their ability to adhere to uroepithelial cells, interaction with macrophages, and for production of virulence factors like extracellular proteinase, elastase, hemolysin, phospholipase C and pyochelin. Biofilm cells showed increased adherence to UECs, which was coupled with reduced uptake and intracellular killing by macrophages. Overall there was a decrease in production of extracellular products by biofilm cells. Comparing the two cell forms for their ability to establish infection in an ascending model of acute pyelonephritis, significant enhancement of renal bacterial load, as well as more pronounced renal pathology developed with biofilm cells.

Effect of combined aspirin and angiotensin-converting enzyme inhibitor therapy versus angiotensin-converting enzyme inhibitor therapy alone on readmission rates in heart failure.

An adverse interaction between aspirin and angiotensin-converting enzyme (ACE) inhibitors is suspected in patients with heart failure, but the effect of combined therapy with these agents on hospital readmission rates is unknown. Our study found that combining aspirin with ACE inhibitors is associated with higher early readmission rates than use of ACE inhibitors alone, particularly in patients with depressed ejection fraction and in those without coronary artery disease.

Simple clinical variables are markers of the propensity for readmission in patients hospitalized with heart failure.

In a consecutive cohort of patients hospitalized for decompensated heart failure, we found that chronic obstructive pulmonary disease and history of hospitalization for any cause in the preceding 6 months were the strongest correlates of early readmission. Based on these findings, we propose a simple risk stratification system to classify patients who are hospitalized for heart failure as low, medium, or high risk for early readmission.

Does left ventricular shape influence clinical outcome in heart failure?

BACKGROUND: Left ventricular (LV) shape tends to become spherical in patients with dilated cardiomyopathy of diverse etiology. Clinical and echocardiographic factors which affect the degree of LV spherical distortion and the impact of altered LV shape on prognosis have not been studied adequately. HYPOTHESIS: This study was undertaken to investigate the prognostic implications of altered LV shape on clinical outcome in dilated cardiomyopathy. METHODS: In 112 patients with depressed LV ejection fraction (19 +/- 9%) and symptomatic heart failure, and in 10 age- and gender-matched normal controls, we performed 2-dimensional echocardiography to assess LV shape using the eccentricity index. Eccentricity index was defined as the ratio of the LV long axis to the LV transverse diameter, measured at end systole and end diastole in the apical four-chamber view. We sought univariate and multivariate clinical and echocardiographic correlates of LV shape. Further, we sought correlations between eccentricity index and clinical outcomes (death and composite outcome of death or emergent heart transplant). RESULTS: Compared with controls, patients with cardiomyopathy had significantly lower systolic (2.04 vs. 1.56; p = 0.001) and diastolic (1.75 vs. 1.53; p = 0.003) eccentricity index, implying a more spherical LV shape. Of all clinical and echocardiographic variables tested, mitral regurgitation, right ventricular dysfunction, and increased LV mass were independently associated with spherical LV shape. At a follow-up period of 17 +/- 12 months, no correlation was found between eccentricity index and the occurrence of death or the combined endpoint of death or emergent heart transplant, in univariate or multivariate analysis. CONCLUSIONS: In patients with dilated cardiomyopathy, the degree of spherical distortion of the LV does not correlate with prognosis.

Pyelonephritic potential of Pseudomonas aeruginosa in ascending mouse model.

The suitability of female BALB/c and Swiss Webster (LACA) strains of mice for an ascending model of pyelonephritis for human uroisolate of Pseudomonas aeruginosa, was assessed. A fresh isolate of Ps. aeruginosa elaborating virulence factors like elastase, protease, phospholipase C, pyochelin and haemolysin was selected and introduced transuretherally in mice without any manipulation of the urinary tract. This isolate was able to colonize and persist in the renal tissue till day 12 post infection. Histopathologically, severe renal lesions i.e., abscesses formation was observed. The LACA strain was found to be more susceptible to infection with the selected Ps. aeruginosa isolate.

Does gender bias exist in the medical management of heart failure?

INTRODUCTION: The purpose of this study was to ascertain the presence of gender bias in the medical management of heart failure, and to assess its association with the specialty of the caregiver physician. METHODS: In 309 patients with documented left ventricular systolic dysfunction (ejection fraction <45%) and at least one hospitalization for heart failure, we assessed the frequency of use of effective medical therapy for heart failure among male (n=187) and female (n=122) patients at the time of hospital discharge. We constructed multivariate models relating patient gender and caregiver specialty to utilization of each class of medications (angiotensin-converting enzyme inhibitors, effective vasodilator therapy (i.e., angiotensin-converting enzyme inhibitors or hydralazine-nitrate therapy), diuretics, digoxin), and combination therapy (i.e., vasodilator plus diuretic plus digoxin). RESULTS: In crude analyses, we did not find any difference in utilization of medications between male and female patients. Multivariate analyses involving adjustment for age, race, coronary artery disease, ejection fraction, and other relevant variables, revealed higher utilization of combination therapy by cardiologists in male versus female patients (adjusted odds ratios=2.07; 95%CI=1.09-3.95), and higher utilization of digoxin therapy by non-cardiologists in female versus male patients (adjusted odds ratio=5.5; 95%CI=1.4-22.2). No gender or caregiver specialty differences were seen in models relating to the other classes of medications. CONCLUSIONS: Our findings suggest the presence of gender bias in the medical management of heart failure, and identify an interesting interaction between caregiver specialty and gender bias.

New paradigms in preventive cardiology: unconventional coronary risk factors.

The 1996 Bethesda Conference acknowledged the following conditions as possible new cardiac risk factors: left ventricular hypertrophy, homocysteine, lipoprotein(a), hypertriglyceridemia, oxidative stress, and fibrinogen. Left ventricular hypertrophy is an independent risk factor for vascular disease, the improvement or progression of which influences subsequent cardiovascular complications. Clinical trials are currently underway to assess potential benefit from lowering homocysteine levels. The role of lipoprotein(a) excess in vascular disease is controversial; its atherogenic potential seems to be neutralized by effective lowering of LDL-cholesterol. Increasing evidence supports the independent role of hypertriglyceridemia in cardiovascular disease and possible clinical benefit from lowering triglyceride levels. Among antioxidant micronutrients, supplementation with vitamin E has been shown to be beneficial in some but not all primary and secondary prevention studies, but data to support use of other antioxidants are much weaker. Preliminary evidence suggests that the reduction of fibrinogen levels in patients with high baseline levels and coronary disease may be beneficial.Despite the potential relation between new risk factors and cardiovascular disease, routine clinical application of these conditions as cardiovascular risk factors would be premature. We first need evidence that these conditions extend prognostic ability beyond conventional risk factors and that modification of these conditions can decrease the risk of cardiovascular events.

Potential new cardiovascular risk factors: left ventricular hypertrophy, homocysteine, lipoprotein(a), triglycerides, oxidative stress, and fibrinogen.

The 1996 Bethesda Conference acknowledged left ventricular hypertrophy, hyperhomocysteinemia, lipoprotein(a) excess, hypertriglyceridemia, oxidative stress, and hyperfibrinogenemia as possible new cardiac risk factors. This review summarizes the current literature that supports these conditions as cardiac risk factors. Left ventricular hypertrophy is an independent risk factor for vascular disease. Improvement or progression of left ventricular hypertrophy influences subsequent cardiovascular complications. Clinical trials are under way to assess the potential benefit of decreasing homocysteine levels. The role of lipoprotein(a) excess in vascular disease is controversial. The atherogenic potential of lipoprotein(a) seems to be neutralized by effective reduction of low-density lipoprotein cholesterol levels. Increasing evidence supports an independent role of hypertriglyceridemia in cardiovascular disease and a possible clinical benefit from decreasing triglyceride levels. Among antioxidant micronutrients, supplementation with vitamin E has been shown to be beneficial in primary and secondary prevention studies. Data supporting the use of other antioxidants are much weaker. Preliminary evidence suggests that reducing fibrinogen levels in patients with high baseline levels and coronary disease may be beneficial. Despite the potential relation between new risk factors and cardiovascular disease, routine clinical application of these conditions as cardiovascular risk factors would be premature. Evidence is needed that these conditions extend prognostic ability beyond conventional risk factors and that modification of these conditions can reduce the risk for cardiovascular events.