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.

Evidence-based Update of Pediatric Dental Restorative Procedures: Preventive Strategies.

BACKGROUND: There has been significant advances in the understanding of preventive restorative procedures regarding the advantages and disadvantages for restorative procedures; the evidence for conservative techniques for deep carious lesions; the effectiveness of pit and fissure sealants; and the evidence for use of resin infiltration techniques. AIM: The intent of this review is to help practitioners use evidence to make decisions regarding preventive restorative dentistry in children and young adolescents. STUDY DESIGN: This evidence-based review appraises the literature, primarily between the years 1995-2013, on preventive restorative strategies. The evidence was graded as to strong evidence, evidence in favor, or expert opinion by consensus of authors Results: The preventive strategy for dental caries includes individualized assessment of disease progression and management with appropriate preventive and restorative therapy. There is strong evidence that restoration of teeth with incomplete caries excavation results in fewer signs and symptoms of pulpal disease than complete excavation. There is strong evidence that sealants should be placed on pit and fissure surfaces judged to be at risk for dental caries, and surfaces that already exhibit incipient, non-cavitated carious lesions. There is evidence in favor for resin infiltration to improve the clinical appearance of white spot lesions. CONCLUSIONS: Substantial evidence exists in the literature regarding the value of preventive dental restorative procedures.

Evidence-based Update of Pediatric Dental Restorative Procedures: Dental Materials.

BACKGROUND: The science of dental materials and restorative care in children and adolescent is constantly evolving, and the ongoing search for ideal restorative materials has led to plethora of research. AIM: To provide an evidence base to assist dental practitioners choose appropriate restorative care for children and adolescents. STUDY DESIGN: This evidence-based review appraises this literature, primarily between the years 1995-2013, for efficacy of dental amalgam, composites, glass ionomer cements, compomers, preformed metal crowns and anterior esthetic restorations. The assessment of evidence for each dental material was based on a strong evidence, evidence in favor, expert opinion, and evidence against by consensus of the authors. RESULTS: There is varying level of evidence for the use of restorative materials like amalgam, composites, glass ionomers, resin-modified glass-ionomers, compomers, stainless steel crowns and anterior crowns for both primary and permanent teeth. CONCLUSIONS: A substantial amount data is available on restorative materials used in pediatric dentistry; however, there exists substantial evidence from systematic reviews and randomized clinical trials and clinicians need to examine and understand the available literature evidence carefully to aid them in clinical decision making.

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.

Thalidomide: an old drug with new action.

Thalidomide is a drug that, since its development, has made history in the world of medicine--having been withdrawn and now has returned with a boom as an anticancer and immunomodulatory drug. However, its mode of action in various diseases (i.e. different types of hematologic malignancies, solid tumors) as well as in various infections (i.e. pneumonia, tuberculosis, HIV infection etc.) and related inflammatory conditions is not well understood. As the immune system plays an important role in the pathogenesis of both infection-related as well as noninfectious (i.e. cancer) inflammatory diseases, much research has been done in the past few years to discover and design better immunomodulatory agents. Such immunomodulatory agents should be able to target the immune system in such a way that host suffers minimum damage and normal function of the immune system remains intact. In the present review an attempt is made to highlight the immunomodulatory action of thalidomide in various pathologic conditions.

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.

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.

Intravenous 2-chloroadenosine protects BALB/c mice from Klebsiella pneumoniae B5055-induced sepsis by modulating the pro-inflammatory immune response.

Sepsis is a severe inflammatory immune response of the host against an infectious agent or its product i.e. lipopolysaccharide (LPS). Therefore, targeting the immune system during sepsis may lower the morbidity and mortality associated with sepsis. The present study shows the protective immunomodulatory action of 2-chloroadenosine (2-CADO) in K. pneumoniae B5055 induced sepsis in male BALB/c mice. Sepsis was induced by implanting the fibrin-thrombin clot containing known amount (10(2)cfu) of Klebsiella pneumoniae B5055 into the peritoneal cavity of mice. 100% mortality with in the 5 post infection days (PIDs) was observed in control group animals. Intravenous 2-CADO (10 microg/kg/day) treatment increased in survival of animals by 70% without significantly (p>0.05) decreasing the blood bacterial load. But a significant (p<0.05) decrease in the level of inflammatory markers (TNF-alpha, il-1alpha, MDA, NO) responsible for sepsis was observed. However, serum il-10 levels were found to be significantly (p<0.05) increased with 2-CADO treatment.

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.

Protective efficacy of Emblica officinalis against Klebsiella pneumoniae induced pneumonia in mice.

BACKGROUND & OBJECTIVES: Emblica officinalis (amla), which is a good source of vitamin C, has been shown to be beneficial due to its immune system enhancing property coupled with its tonifying and antiageing effect. The present study was conducted to evaluate the effect of E. officinalis feeding on the susceptibility of experimental mice to respiratory tract infection induced by Klebsiella pneumoniae. METHODS: The effect of short- (15 days) and long (30 days)-term feeding of amla in mice on the course of K. pneumoniae ATCC43816 infection in lungs was studied, in terms of bacterial colonization, macrophage activity, malondialdehyde (MDA) and nitrite production in broncheoalveolar lavage fluid (BALF). Tumour necrosis factor (TNF)-alpha level in serum was also assessed. RESULTS: Though there was a decrease in bacterial colonization after short-term feeding, it was not significant. On the contrary, the decrease in bacterial load was significant (P < 0.05) on long-term feeding. The operative mechanisms in terms of lipid peroxidation, phagocytosis and nitrite production were studied by estimating their levels in broncheoalveolar lavage fluid (BALF). Maximum decrease in malondialdehyde (MDA) levels and increase in phagocytic activity and nitrite levels on long-term feeding was seen. INTERPRETATION & CONCLUSION: These results suggest that dietary supplementation with amla protects against bacterial colonization of lungs on long-term feeding in experimental model. Further studies need to be conducted to understand the actual mechanism.

The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized.

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.

Antibiotic-induced release of inflammatory mediators from bacteria in experimental Klebsiella pneumoniae-induced sepsis.

In a fibrin-clot model of sepsis, developed in mice, treatment with the antibiotics ceftazidime (Cfz) and ofloxacin (Ofl) caused significant (p < 0.01) release of endotoxin and TNF-alpha after 4.5 h when compared with control (untreated) and amikacin (Ami) treated group. Except for control group, the level of bacteremia declined in all three antibiotic-treated groups. The results suggest that antibiotic therapy, irrespective of the agent used, results in an increase in endotoxin levels in vivo. The amount of endotoxin liberated by Ami was much smaller than with Cfz and Ofl therapy, which makes it an appropriate agent for the treatment of sepsis. An increase in the level of TNF-alpha along with endotoxin is suggestive of increased inflammatory response.

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.

Immunoprotective potential of polysaccharide-tetanus toxoid conjugate in Klebsiella pneumoniae induced lobar pneumonia in rats.

The polysaccharide (PS) derived from K. pneumoniae NCTC 5055 lipopolysaccharide (LPS) was covalently linked to tetanus toxoid by using carbodimide with adipic acid dihydrazide as a spacer molecule. The conjugate was found to be non-toxic and non-pyrogenic at 100 microg dose level. At a similar dose, the conjugate did not elicit any local skin reaction on intradermal preparatory injection in rabbits. The conjugate was immunoprotective as was evident from the decrease in relative colonization of bacteria in lungs of immunized rats as compared to the control animals. Immunization with the conjugate resulted in alveolar macrophage activation in terms of their ability to phagocytose bacteria in vitro.

Establishment of a sepsis model following implantation of Klebsiella pneumoniae-infected fibrin clot into the peritoneal cavity of mice.

Successful establishment of sepsis by entrapping a dose of 150 colony forming units of Klebsiella pneumoniae in a fibrin clot following implantation into the peritoneal cavity of mice is reported. The dose in the fibrin clot gave 50% mortality in mice, spread over a period of one week. All the infected mice showed positive blood culture up to 6 d post-infection; histopathology revealed inflammatory changes in both liver and spleen. Introduction of K. pneumoniae into experimental mice without entrapment in fibrin clot caused no mortality and blood culture remained positive only up to 2 d; histopathology of liver and spleen throughout the period of study showed relatively mild inflammatory changes, which almost cleared during 14 d post-infection. The use of the fibrin-clot model may thus be considered to be useful in studying both the initial and the persisting stage of infection in the peritoneum, whence a slow release of bacteria into the blood takes place which finally leads to sepsis and septicemia.

Contribution of capsular and lipopolysaccharide antigens to the pathogenesis of Klebsiella pneumoniae respiratory tract infection.

The role of Klebsiella pneumoniae K- and O-polysaccharide antigens was determined in a rat model of lobar pneumonia. The induction of experimental infection in rats by wild-type strain, and its lipopolysaccharide- and capsular polysaccharide-deficient mutants was compared. Though the mutant lacking both antigens (K- O-) induced infection it could not successfully establish itself in the rat lung. It caused only mild infection, as compared to the wild type strain (K+ O+) and the strain lacking CPS alone (K- O+). Besides capsular polysaccharide, the lipopolysaccharide antigen was shown to be an important factor in pathogenesis of K. pneumoniae acute respiratory tract infection.

Breast-feeding and alcoholism: the Trotter hypothesis.

OBJECTIVE: The authors' goal was to determine whether early termination of breast-feeding contributes to later alcohol dependence, as proposed more than 200 years ago by the British physician Thomas Trotter. METHOD: In 1959-1961, a multiple-specialty group of physicians studied 9, 182 consecutive deliveries in a Danish hospital, obtaining data about prepartum and postpartum variables. The present study concentrates on perinatal variables obtained from 200 of the original babies who participated in a 30-year high-risk follow-up study of the antecedents of alcoholism. RESULTS: Of the 27 men who were diagnosed as alcohol dependent at age 30, 13 (48%) came from the group weaned from the breast before the age of 3 weeks; only 33 (19%) of the 173 non-alcohol-dependent subjects came from the early weaning group. When challenged by other perinatal variables in a multiple regression analysis, early weaning significantly contributed to the prediction of the severity of alcoholism at age 30. CONCLUSIONS: The data support the hypothesis that early weaning may be associated with a greater risk of alcohol dependence later in life.

TH1 pattern of cytokine secretion by splenic cells from pyelonephritic mice after in-vitro stimulation with hsp-65 of Escherichia coli.

Splenic lymphocytes and peritoneal macrophages from BALB/c mice with Escherichia coli pyelonephritis were obtained at various intervals after infection. These cells were stimulated in vitro with different antigens and cytokine release was assayed in the supernate of the cultured cells. It was observed that both specific antigens such as outer-membrane proteins (OMPs), porins and heat-shock protein-65 (hsp-65), as well as non-specific mitogens such as phytohaemagglutinin (PHA), were able to induce cytokine production by splenic cells from infected mice. Of all these antigens, hsp-65 was found to be the best inducer of cytokine release. In the acute stage of pyelonephritis, the release of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) was found to increase with time; both reached their peak values on the seventh day after infection. The TH1 pattern of cytokine secretion by splenic cells was observed, i.e., IL-2 and IFN-gamma, whereas there was complete absence of IL-4 secretion. In the chronic stage of pyelonephritis, i.e., 150 days after infection, a decrease in the level of IL-2 and IFN-gamma was observed. Peritoneal macrophages released IL-1 on stimulation with hsp-65, which increased with the progression of disease. The possible implications of this study for the disease process are discussed.

Lipopolysaccharide-induced resistance in mice against ascending urinary tract infection with Klebsiella pneumoniae.

Protective effect of the lipopolysaccharide (LPS) antigen of Klebsiella pneumoniae was tested against ascending-mode urinary tract infection in BALB/c and LACA strains of mice. LPS was given by two different routes; LPS was found to be protective (whatever the application route) since colonization with the challenge organism was significantly lower in both cases as compared with unimmunized mice. A maximum decrease in bacterial count in the kidney of LPS-treated animals was observed on challenge after a 4-d treatment.