Recent methods for discovering novel bioactive metabolites, specifically antimicrobial agents, from marine-associated micro-organisms.

Marine micro-organisms are a promising source for novel natural compounds with many medical and biotechnological applications. Here, we demonstrate limitations and recent strategies for investigating the marine microbial community for novel bioactive metabolites, specifically those of antimicrobial potential. These strategies include culture-dependent methods such as modifying the standard culture media, including changing the gelling agent, dissolving vehicle, media supplementation and preparation to access a broader range of bacterial diversity from marine samples. Furthermore, we discuss strategies such as in situ cultivation, dilution-to-extinction cultivation and long-term incubation. We are presenting recent applications of culture-independent methods such as genome mining, proteomics profiling and the application of metagenomics as a novel strategy for structure confirmation in the discovery of the marine micro-organism for novel antimicrobial metabolites. We present this review as a simple guide and a helpful resource for those who seek to enter the challenging field of applied marine microbiology.

Analysis of the beak and feather disease viral genome indicates evidence of multiple introduction events into Saudi Arabia.

Psittacine beak and feather disease (PBFD), caused by beak and feather disease virus (BFDV) is a highly contagious disease in wild and captive psittacine populations and has an almost global presence. However, the BFDV infection in Saudi Arabia remains largely unknown. In the present study, we report the full genome sequence of BFDV strains from Saudi Arabia and its genetic diversity. The complete genome sequences were analyzed for 14 BFDV-infected birds representing 6 psittacine species. The complete genome sequence of BFDV strains was compared with 201 previously reported sequences to evaluate their diversity and possible recombination events, if any. Our analysis revealed that newly sequenced BFDV genomes from Saudi Arabia belonged to six different strains. Phylogenetic analysis suggested that the isolated BFDV genomes were highly recombinant with a high degree of diversity. It is evident from the study that psittacine species in Saudi Arabia are at risk from the spread of BFDV. As per the CITES trade database, about 190,000 parrots have been imported to Saudi Arabia since 1975 over a thousand instances. Presumably, during any of these trade events or unregulated trade of birds has predisposed the introduction of BFDV to Saudi Arabia. Understanding the epidemiology of BFDV is necessitated to address the threat posed by the virus to the psittacine population of Saudi Arabia.

Preparation and characterization of alginate/silver/nicotinamide nanocomposites for treating diabetic wounds.

Silver/Alginate/Nicotinamide nanoparticles composite (Ag/ALG/Nic) was prepared and used for the first time to fabricate wound dressing material. Sodium alginate (ALG) was used as reducing and stabilizing agents for preparation of silver nanoparticles (Ag-NPs). Effect of concentrations of alginate (ALG) on the particle size of silver were studied and confirmed by different techniques like UV/vis spectroscopy, transmission electron microscope (TEM) and dynamic light scattering (DLS). Nonwoven viscous fabrics were used as a carrier for silver/alginate/nanoparticles composite by impregnated the nonwoven fabrics as per the padding-curing technique. Nicotinamide (Nic) as anti-inflammatory drug was entrapped into Ag-NPS/ALG/nonwoven fabrics. Scanning electron microscope and energy dispersive x-ray (SEM-EDX) were used to evaluate the presence of Ag/ALG/Nic nanoparticles composite anchored the nonwoven fabrics. The antibacterial activity of the Ag/ALG/Nic wound dressing material was evaluated against Escherichia coli (E. coli) and Staphylococcus Aureus (St. Aureus). The wound healing and histological studied were evaluated by using burn diabetic rat animals.

Preparation, characterization and antibacterial activity of chitosan-g-poly acrylonitrile/silver nanocomposite.

Chitosan-grafted-poly acrylonitrile silver nanocomposites (Cs-g-PAN/Ag) were prepared via in-situ chemical reduction of Ag ions in graft copolymerization of acrylonitrile onto chitosan. Graft copolymerization process was provided by FTIR and gravimetric methods. UV spectra and TEM images show silver nanoparticles with average 15-20nm dispersed homogeneously in CS-g-PAN/Ag nanocomposite-ray and TGA evident the change in crystallography and thermal stability in consequence of presence Ag nanoparticles. Cs-g-PAN/Ag nanocomposite showed excellent antimicrobial performance towards bacteria such as Escherichia coli and Staphylococcus aureus.

Chemical composition of the stem bark and leaves of Ficus pandurata Hance.

A new compound, 3-O-alpha-L-arabinopyranosyl-4-hydroxybenzoic acid (13), in addition to 16 newly reported compounds: alpha-amyrin acetate (1), beta-amyrone (2), 3beta-acetoxy-20-taraxasten-22-one (3), alpha-amyrin (4), ceryl alcohol (5), stigmasterol (6), beta-sitosterol (7), 2alpha,3alpha-dihydroxy-lup-20(29)-en-28-oate (8), ursolic acid (9), beta-sitosterol-3-O-glucosoide (10), protocatechuic acid (11), betulinic acid (12), quercetin (14), quercetin-3-O-beta-D-glucoside (15), kampferol-3-O-beta-neohesperidoside (16) and rutin (17) were isolated from the stem bark and leaves of Ficus pandurata (Hance) cultivated in Egypt. Identification of these compounds has been established by physical, chemical and spectral data (UV, IR, MS, (1)H- and (13)C-NMR), as well as comparison with authentic samples.

Pycnodysostosis: clinical, radiologic, and endocrine evaluation and linear growth after growth hormone therapy.

Pycnodysostosis is a rare hereditary bone abnormality with an autosomal recessive mode of inheritance. We report the clinical, radiologic, and endocrine status of 8 children with this rare disease. All patients had the characteristic phenotype of the disorder including short stature (8 of 8), increased bone density (7 of 8), separated cranial sutures (8 of 8), large fontanel with delayed closure (8 of 8), obtuse mandibular angle (8 of 8), delayed teeth eruption (8 of 8), enamel hypoplasia (7 of 8), dysplastic acromial ends of the clavicles (6 of 8), frontal bossing (6 of 8), ocular proptosis (8 of 8), and dysplastic nails (8 of 8). Developmental evaluation according to the revised Denever developmental screening showed normal motor, fine motor-adaptive language, and personal social abilities in all the children. All had normal hepatic and renal functions. Serum calcium and phosphorus concentrations were normal. Two children had low serum alkaline phosphatase concentration. Short stature is a characteristic feature of pycnodysostosis. Seven of the 8 children were born short (length standard deviation score [SDS] = -3 to -1.5). Deceleration of linear growth was significant during the first 3 years of life. All the children had height SDS below -3 at the end of their third year of life. Although short stature is a feature of this genetic disorder, defective growth hormone (GH) secretion in response to provocation with clonidine and glucagon was found in 4 of the 8 patients. These 4 patients had pituitary hypoplasia on the magnetic resonance imaging (MRI) of their brain. In addition, 3 of these 4 patients had demyelination of the cerebrum. Patients with pycnodysostosis (n = 8) had low circulating concentrations of insulin-like growth factor-1 (IGF-1) compared with normal age-matched short children with constitutional short stature (CSS). IGF-I increased significantly after injecting GH for 3 days in these patients. Physiologic replacement with GH (18 U/m(2)/week) divided in daily evening doses subcutaneously increased IGF-1 concentration and improved linear growth velocity and height standard deviation scores (HtSDS) in the 4 children with GH deficiency. These data ruled out GH resistance and proved the usefulness of GH therapy in the management of short stature in these patients. In summary, some patients with pycnodysostosis have partial GH deficiency and low IGF-1 concentration. GH therapy markedly increases IGF-I secretion and improves their linear growth. MRI study of the brain including the hypothalamic-pituitary area is recommended in these children because of the high incidence of pituitary hypoplasia and cerebral demyelination.

Minor phenolics from Crinum bulbispermum bulbs.

From the bulbs of Crinum bulbispermum Milne, four new minor compounds were isolated viz. 4-hydroxy-2',4'-dimethoxydihydrochalcone (1), 4,5-methylenedioxy-4'-hydroxy-2-aldehyde[1,1'-biphenyl] (4), hippacine (6), and 4'-hydroxy-7-methoxyflavan-3-ol (7). In addition, four known compounds were isolated and identified as 2(S),3',4'-dihydroxy-7-methoxy flavan (2), isolarrien (3), isoliquiritigenin (5) and liquiritigenin (8). The structures of the isolated compounds were established by spectral evidence.

Intestinal spore-forming protozoa among patients suffering from chronic renal failure.

Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis and Microsporidia are four intestinal spore-forming protozoa that cause diarrhoea in immuno-competent individuals and immuno-suppressed patients. Fresh stool samples were obtained from 120 patients suffering from CRF and attending the Dialysis Unit of Zagazig University Hospital. Also, stool samples were obtained from 40 immuno-competent individuals complaining of diarrhoea (control group). The stool samples were examined by direct smear and formol-ether concentration methods then stained by Giemsa, Modified Ziehl Neelsen (MZN) and Aniline carbol methyl violet stains. The four intestinal spore-forming protozoa were detected in 40/120 (33.3%) of patients with CRF and in 2/40 (5.0%) of the control group with a statistically highly significant difference (P < 0.001). C. parvum, Microsporidia, C. cayetanensis and I. belli were detected in 18/120 (15%), 10/120 (8.3%), 9/120 (7.5%) and 3/120 (2.5%), respectively. The four protozoa were found as mixed infections with other pathogens or as single infections confirming their role alone as a cause of diarrhoea. MZN stain was the most efficient simple, and not expensive.

Different cytokines profiles in spleen cells and liver granuloma of Schistosoma mansoni experimentally infected mice during disease development.

To determine the importance of Th1 and Th2 cells in modulating granuloma formation, mRNA transcripts for Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-5) cytokines were assessed by the molecular technique of in situ hybridization in the liver granuloma. The molecular studies showed few number of cells expressing mRNA transcripts for INF-gamma whereas, considerable number of IL-2 cells were present in the liver granuloma at 6 weeks post-infection (p.i.). Complete disappearance of IFN-gamma expressing cells were found when the disease progressed to 13 weeks p.i. Conversely, very high number of cells expressing mRNA transcripts for IL-4 and fair number of IL-5 cells were present at 7 weeks p.i. with a peak level of IL-4 cells at 13 weeks p.i. These in situ molecular studies of the liver tissues, demonstrated that Th1 cells were present at the very early granuloma development. Moreover, Th2 cells were required for its full development. The main interesting finding was the number of cells expressing mRNA for IL-4, as they were very huge and it might exceed the total number of lymphocytes per se in the granuloma. Lymphocytes from experimentally infected mice-spleen cells were also cultured in vitro with S. mansoni soluble egg antigen (SEA) and the same cytokines of lymphocyte supernatant were measured by ELISA assay. The levels of IFN-gamma and IL-2 were high to 6 wk. p.i. with a slight decline of IFN-gamma, and increasing amount of IL-2 at 10-13 wk p.i. Spleen lymphocytes of fully formed granuloma secreted high levels of IL-4 and IL-5. The results suggest that the development of schistosome egg-induced liver granuloma is a complex process and both Th1 and Th2 cell subsets sharing with other inflammatory cells (non lymphocytes), may play an important role in regulating and modulating the immuno-pathology of granuloma formation and the subsequent hepatic fibrosis.

Pleural lavage: a novel diagnostic approach for diagnosing exudative pleural effusion.

Patients with pleural effusions frequently present a diagnostic and therapeutic challenge. The diagnosis is based on the interpretation of the results of thoracentesis or pleural biopsy. When a malignant tumor metastasizes to the pleura, tumor cells can be seeded over the mesothelial surface or in the subserous layer. In the former situation, tumor cells are abundant in pleural fluid, but in the latter, few malignant cells are exfoliated into the pleural cavity, and microscopic deposits may not be visualized at thoracoscopy. Pleural lavage cytologic study at the time of thoracoscopy has not been studied. The purpose of this study was to assess the value of thoracoscopic pleural lavage as an adjuvant in the diagnostic workup of patients with exudative pleural effusions. Fifty patients with exudative pleural effusions were investigated by pleural fluid cytologic findings, Abram's pleural biopsy, thoracoscopy, and pleural lavage cytologic findings. After aspiration of all pleural fluid, 300 mL saline was instilled into the pleural cavity and then recovered for cytologic analysis. The final diagnoses were 32 malignant (64%), 15 tuberculous (30%), and 3 idiopathic (6%) effusions. In the malignant group, thoracoscopic biopsy had the highest yield (94%) followed by lavage cytologic analysis (84%), fluid cytologic analysis (62%), and biopsy with Abram's needle (50%). The sensitivity of combined thoracoscopy and lavage cytologic analysis was 96%. In the patients with tuberculous pleuritis, the yield from the pathologic examination of the biopsy specimen was 93% with thoracoscopy and 60% with the Abrams needle. The diagnostic yield with cytologic analysis on pleural lavage fluid is significantly higher than that on pleural fluid. This is probably because the cells in the lavage fluid are fresher and better preserved than those in the regular pleural fluid, which may have undergone degenerative changes, yielding false-negative results. Pleural lavage cytologic analysis should be performed in patients with suspected malignant pleural effusion who are subjected to diagnostic thoracoscopy, because it may provide additional information to thoracoscopic biopsy.

The schistosome granuloma: characterization of lymphocyte migration, activation, and cytokine production.

Granuloma formation and its regulation are dependent on lymphocytes. Therefore, we compared the characteristics of lymphocytes derived from the spleens and granulomas of Schistosoma mansoni-infected mice during the course of their disease. We examined lymphocyte cell cycle kinetics, migration, expression of activation Ags (CD69 and IL-2R), cytokine production (IL-2, IL-4, IFN-gamma), and apoptosis. Lymphocytes in the G2/M phase of the cell cycle and high levels of lymphocyte intracellular IL-2 were found in the spleen but not in the granuloma. Cell trafficking experiments showed Ag-specific recruitment of schistosomal egg Ag (SEA)-reactive lymphoblasts into granulomas in vivo, as well as recruitment to, residence within, and egress from granulomas in vitro. Granuloma-derived lymphocytes were more highly activated than splenic lymphocytes based on higher levels of CD69 and IL-2R expression. While the granuloma microenvironment was rich in Th2 cytokines, during peak granuloma formation, the lymphocytes per se from the spleen and granuloma did not exhibit a dominant Th1 or Th2 cytokine profile, producing low but similar levels of IL-4 and IFN-gamma. The discrepancy between high IL-2R expression and low levels of IL-2 protein production by granuloma lymphocytes was associated with increased apoptosis in the granuloma compared with the spleen. These findings support the hypothesis that granulomas may play a role in the regulation of systemic pathology in schistosomiasis by adversely affecting the survival of SEA-reactive, immunopathogenic T lymphocytes.

Inhibition of motility and adherence of Proteus mirabilis to uroepithelial cells by subinhibitory concentrations of amikacin.

The effect of subinhibitory concentrations of amikacin on Proteus mirabilis motility and adherence to human uroepithelial and to HeLa cells was compared with that of gentamicin. In addition, the effect of both antibiotics on cell surface hydrophobicity was also examined. Exposure of bacterial cells in the logarithmic phase to one fourth of amikacin or gentamicin at one fourth of their respective minimal inhibitory concentrations causes the inhibition of swarming and motility of Proteus strains. Amikacin significantly reduced adhesion of Proteus strains to human uroepithelial cells and gentamicin exerts the same effect to a lesser extent. Such inhibitory concentrations of amikacin or gentamicin had no significant effect on the attachment ability of these strains to HeLa cells compared to the nontreated cells. Treatment of the bacterial cells with amikacin or gentamicin changed significantly the cell surface hydrophobicity towards the hydrophilic state compared to nontreated cells, and it was found to be strain dependent. Since motility and attachment ability are considered as pathogenic traits, these data indicate the impact of amikacin on the virulence factors especially in urinary tract infections with Proteus strains.

Comparative efficacy of successive exposure of Pseudomonas aeruginosa to gentamicin and ceftazidime.

Aminoglycoside and beta-lactam antibiotics, when used in combination, are usually given simultaneously, however, successive administration may be more efficient. The killing capacity was used to assess the effect of time intervals between low and high concentrations (2-8xMICs) of gentamicin and/or ceftazidime on Pseudomonas aeruginosa and to determine which drug is better to be administered first. The killing capacity after exposure to the antibiotic for 1 h were compared: (i) cells treated with gentamicin alone; (ii) cells treated with ceftazidime alone; and (iii) ceftazidime was added to (i) or (iv) gentamicin was added to (ii) at 0, 1 and 3 h of antibiotic removal. The bactericidal activity of gentamicin was potentiated and the viable cells decreased up to 6 h after antibiotic removal when the ceftazidime was added at O and at 1 h but the extent of bactericidal activity was reduced, when it was added at 3 h after gentamicin removal. Alternatively, treating the cells first with ceftazidime and then gentamicin was added after drug removal at O and at 1 h resulted in a marked decline in the viable cells, while addition of gentamicin after 3 h from ceftazidime removal, the extent of bactericidal activity was reduced. The non-treated cells with gentamicin started to grow heavily within 6 h of ceftazidime removal. No viable cells were detected after overnight incubation in cultures treated first with 6 or 8xMIC of gentamicin for 0.5 or 1 h. This in vitro study suggests that the optimum interval between gentamicin and ceftazidime doses, which gave the maximum bactericidal effect and the time before re-growth, appeared to be 1-2 h.

Effect of beta-lactamase inhibitors on normal immune capabilities and their interactions with staphylococcal pathogenicity.

The effects of the beta-lactamase inhibitors, clavulanic acid, sulbactam and tazobactam on normal immune responses were investigated. These agents did not interfere with either humoral or cell-mediated immune responses as measured by the hemolytic plaque assay and delayed type hypersensitivity reaction assay respectively. In addition, human polymorphonuclear leukocyte phagocytic activity was not altered by these agents. When these agents were tested for their effect on Staphylococcus aureas adherence to buccal epithelial cells we found that all inhibitors suppressed staphylococcal adherence at therapeutic serum concentrations. Among the inhibitors investigated, sulbactam was found to significantly inhibit the hemolysin production of S. aureus. These data suggest that beta-lactamase inhibitors do not exhibit immunomodulating activity, but they interfere with some of the virulence factors of S. aureus. These findings suggest the advantage of preparations containing these inhibitors.

Suppression of immunopathology in schistosomiasis by interleukin-2-targeted fusion toxin, DAB389IL-2. I. Studies of in vitro and in vivo efficacy.

Schistosomiasis causes pathology in an estimated 200 million individuals. Clinical disease is caused by a complex immunopathologic response to the parasite ova, which are deposited in the host tissues. This immunopathologic response is caused by T lymphocytes which express the high-affinity IL-2 receptor (IL-2R). DAB389IL-2 is a diphtheria toxin-IL-2 fusion toxin protein which functionally inactivates or kills cells which bear the high-affinity IL-2R. DAB389IL-2 has been used in man to suppress IL-2R-dependent immune reactivity. Therefore, we reasoned that DAB389IL-2 might suppress immunopathology in schistosomiasis. In these studies we assessed the in vitro and in vivo effects of DAB389IL-2 on the development of immunopathology in murine schistosomiasis. DAB389IL-2 suppressed IL-2, lectin mitogen (Con A), and soluble Schistosoma mansoni egg antigen-induced lymphocyte proliferation and in vitro granuloma formation. In addition, DA-B389IL-2 suppressed in vitro IL-2R expression. DA-B389IL-2 also suppressed the development of granulomas and collagen deposition in vivo in the livers of infected animals. Therefore, DAB389IL-2 may have potential for the targeted reduction of immunopathology due to schistosomiasis in man.

Determination of meropenem in plasma by high-performance liquid chromatography and a microbiological method.

A rapid and selective high-performance liquid chromatographic (HPLC) method for the quantitative determination of meropenem in plasma is described. The drug was separated from plasma after plasma protein precipitation with 15% of trichloroacetic acid. The mobile phase consisted of acetonitrile-water-glacial acetic acid (21.2, 78 and 0.8% v/v, respectively) delivered at a flow rate of 1.2 ml/min. Meropenem was quantified using ultraviolet detection at 296 nm. Meropenem and the internal standard (pheniramine) were well separated from plasma components. The drug could be assayed by the HPLC method in the presence of its analogue, imipenem. The detection limit in plasma was 25 ng/ml of meropenem. The results were compared with those of agar for a microbiological diffusion method using Escherichia coli ATCC 25922 as the test organism. The sensitivity of the microbiological assay was less than 5 ng/ml, but this decreased at higher concentrations. Both methods were applied to the determination of the drug in aqueous solutions and in plasma.

Post-antibiotic effect of azithromycin and erythromycin on streptococcal susceptibility to phagocytosis.

The effect of azithromycin and erythromycin on growth, cell surface hydrophobicity and the susceptibility to the bactericidal activity of human polymorphonuclear leucocytes (PMNL) was examined in four Streptococcus species. Exposure to either 10 x MIC azithromycin or erythromycin induced a post-antibiotic effect (PAE) of between 2.4 and 4.3 h. Erythromycin caused a longer PAE for S. sanguis than azithromycin under the same conditions. The cell surface charge (hydrophobic or hydrophilic) of the streptococci was altered significantly during PAE; loss of hydrophobicity was induced by both macrolides, and this effect was variable amongst the species. The decrease in hydrophobicity was not related to inhibition of growth. The effect of each drug during PAE on the interaction of opsonised suspensions of the streptococci with human PMNL revealed that erythromycin, and to a lesser extent azithromycin, increased susceptibility to the bactericidal activity of human PMNL; this effect was abolished following PAE. The present study clearly showed that PAE should not only be considered as delayed bacterial growth, but also as modulation of bacterial susceptibility to phagocytosis which may influence the outcome of the host-parasite relationship.

Effect of beta-lactamase expression on susceptibility of local isolates of Enterobacter cloacae, Serratia marcescens and Pseudomonas aeruginosa to beta-lactam antibiotics.

During surveillance studies, a total of 66 strains of gram-negative bacilli (28 Enterobacter cloacae, 20 Serratia marcescens and 18 Pseudomonas aeruginosa) with a reduced susceptibility to third-generation cephalosporins, aztreonam and amikacin were isolated from documented infections. All isolates were highly susceptible to imipenem and sparfloxacin. beta-Lactamase activity was demonstrated in all isolates of E. cloacae and S. marcescens, and in 77% of P. aeruginosa isolates. Inducible beta-lactamase activity was detected in 80, 65 and 40% of E. cloacae, S. marcescens and P. aeruginosa isolates, respectively, when cefoxitin was used as an inducer. More inducible beta-lactamase producers were observed when imipenem was used as an inducer. Isolates of E. cloacae, and to a lesser extent S. marcescens, expressed a wide spectrum of beta-lactamase activities. There was a good correlation between baseline beta-lactamase activity and the respective MIC of ceftazidime, cefotaxime and ceftriaxone, and to a lesser extent aztreonam in E. cloacae and S. marcescens isolates. Only one isolate of E. cloacae demonstrated an extended beta-lactamase activity. The data suggest that the resistance of E. cloacae and S. marcescens isolates to beta-lactam antibiotics is largely dependent upon hyperproduction of beta-lactamase.

In vitro activity of subinhibitory concentrations of quinolones on urea-splitting bacteria: effect on urease activity and on cell surface hydrophobicity.

The effect of subinhibitory concentrations of ciprofloxacin, lomefloxacin, norfloxacin, ofloxacin, and sparfloxacin on urease activity and on cell surface hydrophobicity of urea-splitting bacteria was examined. Quinolones at 0.5 MICs demonstrated variable effects on bacterial-urease activity. Norfloxacin inhibited enzyme activity in Proteus vulgaris and Proteus mirabilis, while other quinolones had no effects. In Morganella morganii, sparfloxacin and ciprofloxacin enhanced urease activity, particularly at the initial phase of growth. All quinolones tested showed no marked effect on urease activity by Providencia rettgeri. Quinolones at the same concentrations induced an increase in the cell surface hydrophobicity, which was strain-dependent. There was no correlation between urease inhibition and cell surface hydrophobicity. Inhibition of urease activity by quinolones, in addition to their antibacterial activities, may prevent the progression of urinary tissue damage and stone formation.