In vitro evaluation of vincristine and fluconazole combination against Candida.

Infections associated with cancer are a major scourge and cause of substantial morbidity and mortality in cancer patients. The aim of present study was to appraise the in vitro activity of anticancer agent vincristine and antifungal fluconazole alone and in combination against Candida spp. Results were interpreted in terms of fractional inhibitory concentration index (FICI). Antifungal activity of fluconazole showed marked synergism when used in combination with vincristine, with FICI ranging from 0.25-0.5 against different Candida spp. Although, the use of vincristine with fluconazole is always disputed due to its side effects including decreased peristalsis, but the present research can help to perform suitability analysis of fluconazole use in life threatening invasive candidiasis associated with cancer patients. In addition, the synergism in antifungal activity after using with vincristine also warrants further research in the direction of minimizing adverse reaction associated with combined use of fluconazole and vincristine.

Elucidation of antibiotic effectiveness against Staphylococcus epidermidis during intraocular lens implantation.

The effect of various antimicrobial agents commonly used in irrigating solutions on the hydrophobicity and adherence of Staphylococcus epidermidis ATCC 14990 was investigated. The longest post-antibiotic effect (PAE=3.2 h) was obtained with gentamicin followed by ciprofloxacin (2.7 h), clindamycin (2.2 h), ceftazidime (1.8 h) and vancomycin (1.6 h). The post-antibiotic effect on surface hydrophobicity of cells previously treated with gentamicin, ciprofloxacin, or clindamycin for 120 min resulted in a substantial decrease in affinities to hexadecane (31.4, 28.5 and 27%, respectively) compared with control untreated cells. Less effect was noted with cells previously treated with ceftazidime or vancomycin (20.5 and 15.8%, respectively). Similar but less marked results were obtained when the cells were exposed to antibiotics for 30 or 60 min. The post-antibiotic effect on adherence of cells to both intraocular lenses and to epithelial cells showed that adherence to lenses decreased as the time of exposure to antimicrobial agents increased. Adherence was greatly diminished with cells treated with gentamicin or ciprofloxacin compared with control untreated cells. Adherence was less affected by clindamycin, ceftazidime and vancomycin. The data supported the use of antimicrobial agents in irrigating solution during intraocular surgery, since, reduced adherence (colonization) and might result in a lower incidence of endophthalmitis.

Plasmid profile and antibiotic resistance in coagulase-negative staphylococci isolated from polluted water.

Four different species of coagulase-negative staphylococci (CNS) were isolated from polluted waters in Fez, Morocco and found to be Staphylococcus simulans, Staph. lenticus, Staph. hyicus and Staph. xylosus. Eight isolates belonging to these four species were analysed for their plasmid content. Southern blot hybridizations were performed to define the resistance determinants of the plasmids harboured by these species. These determinants were found to be carried mainly by Class I staphylococcal plasmids (1-5 kb). A plasmid (4.3 kb) carrying a tetracycline resistance gene was present in five isolates from all identified species. Plasmids carrying a chloramphenicol resistance gene were more frequently encountered and found to be of different sizes. Plasmids carrying erythromycin, neomycin, and streptomycin resistance genes were less frequent and were the same size. The results indicate that the occurrence of multi-resistant CNS in polluted waters may constitute a reservoir for disseminating antibiotic-resistance into the community.

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.

Antimicrobial resistance and prevalence of extended spectrum beta-lactamase among clinical isolates of gram-negative bacteria in Riyadh.

The activity of ciprofloxacin, imipenem and 12 other commonly used antibiotics was evaluated against 106 documented clinical isolates from a medical Intensive Care Unit (ICU). The resistance rates to ceftriaxone, cefotaxime, aztreonam and ceftazidime were 42, 25, 24 and 21%, respectively. Apart from Pseudomonas aeruginosa, all isolates were sensitive to ciprofloxacin and imipenem. Complete cross resistance among tested beta-lactam groups was uniformly evident in Enterobacter cloacae, Citrobacter freundii and P. aeruginosa. On the other hand, penicillins and second generation cephalosporins showed cross resistance among Escherichia coli and Klebseilla pneumoniae isolates. Induction experiments indicate that 70 and 62% of P. aeruginosa and E. cloacae or C. freundii produce class I cephalosporinase, respectively. Among all tested isolates, plasmid mediated extended spectrum beta-lactamase (ESBL) was detected in one isolate of K. pneumoniae. The plasmid mediated beta-lactamase is transferable and inhibited by beta-lactamase inhibitors. The transconjugates not only expressed resistance to extended spectrum beta-lactams and aztreonam but also toward tested aminoglycoside antibiotics, with the exception of gentamicin. The obtained transconjugates conferred high level resistance to ceftazidime and aztreonam but considerably low resistance to ceftriaxone and cefotaxime. The isoelectric point for the extended-spectrum beta-lactamase is 8.2.

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.

Differential inhibition by clindamycin on slime formation, adherence to teflon catheters and hemolysin production by Staphylococcus epidermidis.

Slime formation was detected in Staphylococcus epidermidis strains isolated from either infected patients or healthy individuals. Cells of S. epidermidis, that either formed slime or not, adhered to teflon catheters. There was no correlation between adherence of bacteria to teflon catheters and slime formation. Clindamycin at subinhibitory concentration significantly inhibited slime formation without inhibiting bacterial growth. Adherence of S. epidermidis to teflon catheters was affected by the presence of clindamycin whether slime was produced or not. Clindamycin at subinhibitory concentrations markedly inhibited hemolysin production by S. epidermidis without appreciably altering the cell density, and cells grown in the presence of the drug showed very low hemolytic activity upon disruption. These results suggest that clindamycin at low concentration alters S. epidermidis virulence properties, apart from inhibiting growth.

Processing of envelope polypeptides of herpes simplex virus type 1. Demonstration of variation in different cell lines by high-performance liquid chromatography and radioimmunoprecipitation.

[35S]Methionine-labelled envelope polypeptides of herpes simplex virus type 1, strain F, propagated in mammalian cell culture of various origins, were separated by ion-exchange high-performance liquid chromatography on a TSK DEAE-3SW column. Analysis of the fractions by radioimmunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of the immunoprecipitates showed similarities as well as distinct differences in the number, migration patterns and molecular mass of the synthesized polypeptides, depending on the host cell. The results show that this method can be used to demonstrate species-specific or organ-specific differences in the processing of virus-specified polypeptides synthesized in host cells.

Changes in free amino acids in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes after treatment with diazepam.

The effect of single and chronic (15 days) i.p. injections (1.0 and 8.0 mg/kg) of diazepam (DZ) on free amino acid profile in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes of male Wistar Albino rats were investigated. Depletion of some free amino acids was observed in the lymphocytes (mixed T- and B-lymphocytes) and PMN leukocytes (91-95%) neutrophils especially after chronic DZ-treatment. A dose-dependent depletion in the lymphocyte amino acids, Tau, Gly, Ala, Met and Ile, was found after both acute and chronic DZ-treatment. A similar depletion of Tau, Asp, Glu and Met appeared in the PMN leukocytes after single doses as well as chronic DZ-treatment. These results suggest that administration of 1.0-8.0 mg/kg of DZ in single dose or after chronic administration may interfere with the transport of certain important amino acids and/or protein turnover in PB lymphocytes and PMN leukocytes. On the other hand, the basic amino acids Lys, His and Arg were significantly increased in PMN leukocytes after chronic administration of 1.0 mg/kg DZ. It was suggested that the increased levels of the basic amino acids in the neutrophils may interact with the intracellular changes in pH that normally accompany the respiratory burst.

Effect of oral activated charcoal on vancomycin clearance in rabbits with acute renal failure.

The effect of oral administration of activated charcoal on total body clearance of vancomycin administered intravenously (7.5 mg/kg) has been studied in normal rabbits and rabbits with induced renal failure. Gastric intubation of a single dose (10 g) of activated charcoal to normal rabbits did not produce a statistically significant effect on any pharmacokinetic parameter for vancomycin. The mean vancomycin clearances were (mean +/- s.d.) 80.82 +/- 6.8 and 75.24 +/- 9.61 ml/h/kg with and without activated charcoal administration, respectively. To examine whether renal failure would influence the effect of activated charcoal and enhance the systemic clearance of vancomycin, uranyl nitrate was used (0.75 mg/kg, i.v.) to induce acute renal failure in rabbits. The derived pharmacokinetic parameters of vancomycin were consistent with renal failure. No significant differences were observed in any of the calculated pharmacokinetic parameters between the control and charcoal treated rabbits. The lack of effect may be attributed to the large molecular weight of vancomycin.

Determination of lomefloxacin in biological fluids by high-performance liquid chromatography and a microbiological method.

A high-performance liquid chromatographic method (HPLC) was developed for the determination of lomefloxacin in plasma and urine and was compared to a microbiological assay. Lomefloxacin and norfloxacin (internal standard) were extracted from plasma and urine samples using chloroform. Measurements were carried out with a fluorescence detector using an excitation wavelength of 280 nm and an emission wavelength of 430 nm with a mercury lamp. Quantification was achieved by the measurement of the peak-height ratio and the analytical recovery of the drug from plasma and urine was found to be (mean +/- SD) 99.3 +/- 3.74% and 95.7% +/- 3.82%, respectively. In the microbiological assay, E. coli ATCC 1346 was the test organism using an agar diffusion technique. The coefficients of variation for within-day analysis for both the HPLC method and microbiological assay from plasma samples were less than 7%. The minimum detectable concentration for both the HPLC and the microbiological method was 50 ng/ml and 100 ng/ml, respectively. Both methods were used to determine the lomefloxacin level in plasma following intravenous administration to mice. Excellent agreement was obtained between the results of the two methods. The HPLC method offers significant advantages in accuracy, precision, speed of analysis and turnover-time.

A nitrate reductase-based colorimetric assay for the study of bacterial adherence.

Washed intact cells of Escherichia coli and Staphylococcus aureus, grown under partial anaerobic conditions in nitrate media, reduced nitrate quantitatively when formate was used as a reducing substrate. Nitrate reductase was applied as an index for bacterial adherence to different target surfaces including uroepithelial cells, HeLa cells and fibrin clots. Nitrate reduction by adhered as well as control cells was determined by quantitative diazotization reaction for nitrite. Variations in the conditions which affect adherence gave rise to corresponding variations in the nitrate reduction index from which bacterial adherence can be conveniently determined under these conditions. This method is simple, reproducible and easy to perform in a short time.

Effects of vancomycin, teicoplanin, daptomycin and coumermycin on normal immune capabilities.

Vancomycin, teicoplanin, daptomycin, and coumermycin were investigated for immunomodulatory activity on both humoral and cell-mediated immune responses in Balb/c mice. All four antibiotics did not produce any interference with these responses as measured by hemolytic plaque assay and delayed-type hypersensitivity to sheep red blood cells. The administration of these antibiotics for seven days did not affect the peripheral blood leukocyte count or spleen weights. When these antibiotics were tested for their interaction with human polymorphonuclear phagocytic activity, no alteration in this activity was observed. These findings suggest that vancomycin, teicoplanin, daptomycin, and coumermycin may be safely used for the treatment of infections without altering host-defence mechanisms.

Antimicrobial activity of artemisinin and its derivatives against anaerobic bacteria.

Artemisinin and nine of its semisynthetic derivatives were tested for antibacterial activity against anaerobic, facultative anaerobic, microaerophilic and aerobic bacteria. Only anaerobic bacteria and gonococci showed sensitivity to artemisinin derivatives. Artemisinin and its deoxy derivatives had no activity at 100 micrograms/ml concentration. The newly synthesized methyl diperoxy derivative had the highest activity against all tested anaerobes with a MIC of 9.4 micrograms/ml.