Biomedical subjects
Publications and source records attributed to F Eftekhar.
Phytochemical investigation of the choloroform extract of Salvia leriaefolia afforded 8(17),12E,14-labdatrien-6,19-olide (1), and its structure was determined by a combination of spectral methods. Compound 1 was found to possess antibacterial activity against Staphylococcus aureus.
The exopolysaccharide (alginate) of mucoid strains of Pseudomonas aeruginosa is believed to be an important virulence factor. The ability of an alginate-deploymerizing enzyme (alginase) to modify the polymorphonuclear leukocyte (PMN)-directed and antibiotic-mediated phagocytosis and killing of mucoid P. aeruginosa was studied both in vitro and in vivo. In vitro, pretreatment of a mucoid P. aeruginosa strain (144MR) resulted in a significant enhancement of PMN phagocytosis and killing of the organism (P less than 0.05), to levels similar to that observed with its nonmucoid mate, strain 144NM. Moreover, alginase treatment of the mucoid strain 144MR caused a substantial removal of bacterial cell surface alginate as assessed by immunofluorescence staining with a murine monoclonal antialginate antibody. The experimental endocarditis model was used to evaluate the in vivo effect of alginase in modifying the course of a deep-seated pseudomonal infection caused by mucoid strain 144MR. In right-sided endocarditis, in which PMNs normally mediate spontaneous clearance of the organism from cardiac vegetations (A. S. Bayer, J. Yih, C. Y. Chiu, and C. C. Nast, Chemotherapy 35:278-288, 1989), the presence of the alginate exopolysaccharide on strain 144MR was associated with an inability to reduce intravegetation pseudomonal counts over a 13-day postinfection period; in contrast, right-sided vegetations infected with the nonmucoid strain 144NM underwent significant reductions in bacterial densities over this same time (P less than 0.05). Administration of alginase intravenously (i.v.) (750 enzyme units per day for 7 days) to animals with right-sided endocarditis caused by the mucoid strain 144MR was associated with a significant reduction in intravegetation pseudomonal counts (P less than 0.05), to levels similar to that seen with endocarditis caused by the nonmucoid strain. In left-sided endocarditis caused by mucoid strain 144MR, animals received either no therapy, amikacin (20 or 40 mg/kg twice a day for 7 or 14 days), or amikacin plus alginase (750 U/day [i.v.]). The coadministration of alginase for 14 days with the higher-dose amikacin regimen rendered more left-sided vegetations culture negative than those in animals receiving the antibiotic alone for 7 or 14 days (P = 0.001 and 0.056, respectively). These salutary effects of alginase in vivo were paralleled by the ability of the enzyme to remove the exopolysaccharide from the surface of mucoid pseudomonal cells within cardiac vegetations, as assessed by transmission electron microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)
We previously showed substantial differences in Pseudomonas aeruginosa exopolysaccharide production in vitro at oxygen tensions reflective of the right versus left cardiac circuits in vivo (40 versus 80 mm Hg, respectively; A. S. Bayer, T. O'Brien, D. C. Norman, and C. C. Nast, J. Antimicrob. Chemother. 23:21-35, 1989). However, those studies did not specifically confirm this exopolysaccharide to be the characteristic P. aeruginosa mucoid alginate seen in patients with cystic fibrosis. With a murine monoclonal antibody prepared against P. aeruginosa alginate, strongly positive immunofluorescence (IF) staining of a nonmucoid P. aeruginosa strain (PA-96) was seen after its exposure in vitro to oxygen tensions (pO2) of approximately 80 mm Hg; the intensity of the IF staining under these conditions was similar to that observed with a phenotypically mucoid P. aeruginosa strain (C1712M) from a cystic fibrosis patient. In contrast, the same nonmucoid strain (PA-96), after exposure to pO2 of approximately 40 mm Hg, showed little IF staining for alginate. Following enzyme treatment with alginase, PA-96 cells previously exposed to the higher pO2 and exhibiting enhanced alginate production, as determined by IF staining, now showed no IF staining. Moreover, treatment of the oxygen-up-regulated PA-96 cells with alginase released amounts of unsaturated alginate breakdown products (uronic acids) quantitatively similar to those released by typically mucoid strains treated with the same enzyme. These data indicated that the P. aeruginosa exopolysaccharide in our studies was, indeed, mucoid alginate and that variations in oxygen tensions represent one of the trigger mechanisms for the up-regulation of mucoid exopolysaccharide production.
Mucoid Pseudomonas aeruginosa colonizes and infects the respiratory tract of most older patients with cystic fibrosis. These bacteria resist both opsonin-dependent and -independent phagocytosis by human polymorphonuclear leukocytes and monocyte-derived macrophages. Resistance to phagocytosis is thought to be mediated in part by the mucoid exopolysaccharide associated with the bacterial surface. The purpose of this study was to determine whether degradation of the mucoid exopolysaccharide by alginase enhances bacterial susceptibility to nonopsonic phagocytosis by macrophages. Eight phagocytosis-resistant mucoid P. aeruginosa isolates from patients with cystic fibrosis were studied. The bacteria were treated with a preparation of alginase from Bacillus circulans, and phagocytosis by macrophages was measured by a visual inspection assay. Alginase degradation of mucoid exopolysaccharide was measured by the periodate-thiobarbituric acid assay and by indirect immunofluorescence with a mouse monoclonal antibody to the mucoid exopolysaccharide. Alginase degraded the mucoid exopolysaccharide of all eight mucoid strains tested. Phagocytosis was enhanced in five of the eight strains. Alginase-enhanced phagocytosis was magnesium dependent and heat labile. Alginase may be a useful tool for studying the biological properties of P. aeruginosa mucoid exopolysaccharide.
Five isolates of Pseudomonas aeruginosa (CD2, CD3, CD4, CD5, and CD10) from a patient with cystic fibrosis were examined with regard to several genotypic and phenotypic characteristics to determine whether the patient was colonized with one or several distinct strains. Isolates CD2, CD3, and CD4 were obtained from a single sputum sample, and CD5 and CD10 were obtained 1 and 2 years later, respectively. On the basis of colonial morphology, serotyping, and antibiograms, the five isolates appeared to be different strains. However, Southern blot analysis with a 1.2-kilobase DNA probe containing the P. aeruginosa PAK pilin gene indicated that all five strains were identical at that genetic locus. The pilin genes of the five isolates were cloned and sequenced at the nucleotide level and found to be identical. Southern blot analysis with a probe from a separate region of the P. aeruginosa chromosome, a 741-base-pair PstI-NruI DNA fragment adjacent to the exotoxin A gene, also revealed genetic identity among these five clinical isolates. On this basis, it was concluded that this patient was colonized with a single strain of P. aeruginosa and that the strain had remained genetically stable over a period of 2 years. The predicted pilin sequence of the CD isolates was almost identical to that of strain PA103 (97% homology) and serologically related to PAO pilin, with which it shared 80% homology. No immunological cross-reactivity was detected between the CD and PAK pilins, which shared the least homology (62%) among the four pilins considered in this study. Although all five CD isolates contained identical pilin genes, three had acquired mutations which prevented normal expression of the pilus system. CD3 was a putative regulatory mutant which was unable to produce normal amounts of pilin, and CD4 and CD10 were putative assembly mutants which produced normal amounts of pilin but were unable to assemble the pilin subunit into intact pili.
Three nonmucoid revertant P. aeruginosa strains from cystic fibrosis patients were phagocytized by human polymorphonuclear leukocytes in the absence of serum. Phagocytosis was inhibited by D-mannose and by mannose-containing sugars. Bacteria killed by heat or ultraviolet irradiation or grown in shaken broth were devoid of pili and resistant to nonopsonic phagocytosis. The mucoid parents of two phagocytosis-susceptible nonmucoid revertant strains were resistant to nonopsonic phagocytosis. The nonmucoid revertants were more hydrophobic in nature than the mucoid parents, but they were comparably piliated. Nonopsonic phagocytosis of P. aeruginosa by human neutrophils appears to be mediated in part by pili. Other factors such as the mucoid coating of certain strains may interfere with this process.
Pseudomonas aeruginosa is the predominant respiratory pathogen in patients with cystic fibrosis, but its mechanism of persisting in pulmonary secretions is poorly understood. We observed that three nonmucoid cystic fibrosis P. aeruginosa strains were phagocytized and one strain resisted phagocytosis by human polymorphonuclear leukocytes in the absence of serum. Phagocytosis was assessed by luminol-enhanced chemiluminescence, inspection of stained smears, bactericidal assay, reduction of nitroblue tetrazolium dye, and electron microscopy. Phagocytosis, determined by visual inspection, occurred at 35 degrees C but not at 4 degrees C. Nonopsonic phagocytosis was inhibited most efficiently by D-mannose, mannose-containing saccharides, and D-fructose. Opsonin-dependent phagocytosis of P. aeruginosa and of zymosan was not markedly inhibited by mannose, suggesting different leukocyte receptors for nonopsonic and opsonic phagocytosis.
Synergy between a 2:1 combination of ampicillin and amdinocillin was studied with 10 urinary isolates of Enterobacteriaceae. Synergy was observed with six organisms in a bladder model that reproduced many features of the milieu of the human bladder. Antibiotic susceptibility of these organisms was also determined by the agar incorporation plate method, by tube dilution (urine and broth), microtiter method (urine and broth) and disk susceptibility test. Minimal inhibitory concentrations of individual antibiotics were of no value in predicting the synergistic response and were sometimes misleading. None of the laboratory tests could predict synergy in the bladder model. Evidence is available that the bladder model may be superior to other in vitro tests for synergy studies.
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Disk susceptibility tests for mecillinam were carried out on 167 hospital isolates of Enterobacteriaceae from Vancouver. Inhibition zone sizes with eight experimental variations were compared by three-way analysis of variance. The Association of Clinical Pathologists' method gave significantly (p = 0.0002) larger zones than the Kirby Bauer method (24.9 +/- SD 7.5 and 23.4 +/- 7.3 mm, respectively). 10 microgram mecillinam disks from Mast Laboratories gave significantly (p = 0.0004) larger zones than the Baltimore Biological Laboratories (BBL) equivalent (24.9 +/- 7.6 and 23.4 +/- 7.2 mm, respectively). Differences between Oxoid Iso-sensitest agar and BBL Mueller Hinton agar were not quite significant (24.5 +/- 7.9 and 23.8 +/- 6.9 mm, respectively, p = 0.061). Each disk test method was significantly different from at least one other (Duncan multiple range test, alpha = 0.05). A semilogarithmic regression was established between the antibiotic disk inhibition zone and the MIC for each experimental variation. BBL Mueller Hinton and Oxoid Iso-sensitest media did not give significantly different results in MIC determinants by a surface inoculation method. An additional 146 organisms from hospitals in New York provided a wider range of organisms from a different location. Pooled data from both centres were used to identify breakpoints for mecillinam in the disk susceptibility test using the Kirby Bauer method with BBL disks and media. A 16-mm breakpoint with 10 microgram mecillinam disks provides a realistic and conservative prediction of probable response to attainable serum concentrations of this antibiotic.
The mean minimum generation time in shake culture in urine of 6 urinary isolates of Escherichia coli (21.7 +/- 0.6 min) was significantly shorter (P = 0.0003) than that of 14 isolates of less common urinary pathogens (46.0 +/- 18.6 min). Mixed populations of approximately equal numbers of E. coli cells paired with other urinary, fecal, and urethral organisms were introduced into a laboratory model of the lower human urinary tract. This model used urine as a medium and reproduced some features of the balance between bacterial growth and the flushing effect of urine. After 24 h E. coli formed greater than or equal to 99% of the bacterial population in the bladder model for 16 our of 18 pairs of isolates examined. Relatively high oxygen tensions in urine sample from 18 healthy women (10.9 +/- 22. kPA) and 18 infected patients (8.0 +/- 4.3 kPa) may explain why anaerobic urinary infections are uncommon. The rapid growth rate of E. coli may be one explanation why it is the commonest cause of urinary infection even though it is relatively uncommon at the urethral meatus.
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