Vol. 46, February, 2014.

1/46 MOLECULAR CHARACTERIZATION OF SOME SELECTED ENTEROCOCCUS CLINICAL ISOLATES WITH VARYING RESISTANCE TO GENTAMICIN, RIFAMPICIN AND SOME GLYCOPEPTIDES

Nourhana H. Fanaki, Hoda M.G. Omar*, Nihal H. Moussa* and Eva Edward*

Pharmaceutical Microbiology Department, Faculty of Pharmacy, Alexandria University.

The efficacy of the combination of selected glycopeptides (vancomycin and teicoplanin) with gentamicin or rifampicin, as well as the combination of gentamicin with rifampicin was determined against selected enterococcus clinical isolates. All isolates were screened for their susceptibility to the four tested antibiotics using the antibiotic disc diffusion and the agar dilution methods. The broth dilution methods and the standard E-test were carried out for selected isolates. Antibiotic combinations were tested using the checkerboard technique and the time-kill assay. Vancomycin and teicoplanin showed the highest activity against all the isolates with only 3 and 4 resistant isolates to vancomycin and teicoplanin, respectively. Concerning the E-test, our results showed that this method was characterized by a high specificity, but by somewhat low sensitivity. In General, the studied antimicrobial combinations proved an advantage over the single drug treatment, especially when tested during the log phase. Attempts were done to curethe R-plasmids responsible for gentamicin resistance from selected clinical isolates, using ethidium bromide, also to study the transfer of gentamicin resistance plasmids by conjugation, using the broth mating technique. The curing was successful in 4 out of 5 High Level Aminoglycoside Resistance (HLAR) Enterococcus faecium clinical isolates, with curing rates ranging between 1.5 and 7.5%. Whereas for the transferability of gentamicin resistance plasmids by conjugation, promising conjugation frequencies were obtained ranging from 7.67 X 10-4 to 4.48 X 10-2 CFU / donor cells. The plasmid conferring gentamicin resistance was isolated and transformed to chemically competent cells E. coli DH5α, with a transformation efficiency equal to 44.62 transformants/ µg plasmid DNA. On the other hand, attempts were done to investigate the molecular distribution of genes conferring resistance to the tested antibiotics, if present, among selected isolates. Concerning van genes, van A was only detected in three out of the six tested isolates. However, van B was not detected in any of the tested isolates. van A gene was usually detected in enterococcus isolates showing resistance to both vancomycin and teicoplanin. Of the virulence factors' genes, the gelE, esp and hyl showed similar predominance where each existed in three out of the six tested isolates, while asa1 was the least predominant virulence factors' gene. It was concluded that resistance to glycopeptides (vancomycin and teicoplanin) was not widely spread in Egypt. The rifampicin resistance genes RPOB1 and RPOB2 were detected in 11.76 and 47.06% of the tested isolates, respectively. Multiple and various mutations in such genes could mediate resistance to rifampicin in enterococci. The gentamicin resistance gene, aph(2'')-Ic, was not detected in any of the tested 10 HLAR E. faecium clinical isolates. Resistance to gentamicin, in enterococci, could be chromosomally or plasmid mediated. The presence of genes encoding for gentamicin resistance on plasmids or transposons could facilitate the wide spread of such resistance on larger scales.

2/46 CHARACTERIZATION, KEY FACTORS GOVERNING THE MYCOSYNTHESIS OF AGNPS   AND THEIR EVALUATION AS ANTIVIRAL AGENT

Eman M.A. El-Taher

The Regional Center for Mycology and Biotechnology,

Al-Azhar University, Cairo, Egypt

Nano-biotechnology is an offspring of nanotechnology that has emerged at the interface of nanotechnology and biology.The biosynthesis of nanoparticles has received increasing attention due to the growing need to develop safe, cost-effective and environment friendly technologies for nano-materials synthesis. Here, it is a report on extracellular synthesis method for the preparation of Ag nanoparticles (AgNPs) in water using the extract of Agaricus bisporus, a naturally occurring edible mushroom in Egypt, as reducing and protecting agents. The bio-reduced AgNPs was monitored by ultraviolet-visible spectroscopy and the obtained nanoparticles were characterized by transmission electron microscopy (TEM). Energydispersive x-ray (EDX) analysis indicates that particles are crystalline in nature, and they are confirmed to be crystalline byx-ray diffraction (XRD) patterns. Fourier transform infrared (FTIR) measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles.In this study,thereduced nicotinamide adenine dinucleotide (NADH) was found to be an important reducing agent for the biosynthesis of AgNPs and their formation might be an enzyme mediated extracellular reaction process using other low and high molecular weight substances. The antiviral activity of AgNPs was evaluated on Tomato mosaic virus and it was found to be susceptible to AgNPs.

3/46 THE EFFECT OF DIFFERENT MACROLIDES ON BIOFILM FORMATION AND ERADICATION OF FORMED BIOFILMS BY PSEUDOMONAS AERUGINOSA

Mostafa A. El Nakeeb, Nadia M. El-Guink, Hamida M. Abou Shleib

and Nihal K. Moussa

Department of Pharmaceutical Microbiology, Faculty of Pharmacy,

Alexandria University, Egypt.

Pseudomonas aeruginosa is one of the important organisms producing different virulence factors. One of the most important virulence factors affecting the progress of treatment is biofilm formation. Macrolides are potential agents affecting both formation and eradication of biofilm. In the present work, the effect of different concentrations; sub-inhibitory and lethal   concentrations, of different macrolides: azithromycin, clarithromycin, erythromycin, spiramicin, roxithromycin and the ketolide telithromycin on the ability of ten Ps. aeruginosa strains; one standard strain and nine clinical isolates, on both formation and eradication of biofilm was tested. The effect of subinhibitory concentrations; ¼ MIC, of different macrolides on biofilm formation was strain dependent. During biofilm synthesis, variable effects were noted, the results varied between minor and potential effects; 0.14 and 2.25 log decrease in the number of survivors respectively. Among the tested strains, the standard strain Pst was highly sensitive to the intervention done to prevent biofilm formation, as the average log decrease in survivors was 2.25, followed by the clinical isolate P10 with 1.5 log decrease in the number of cells. The clinical isolate P5 was almost unaffected by the presence of marolides. The effect of macrolides on the other strains was rather slight; the average log decrease in survivors varied between 0.14 and 0.7. Roxithromycin possessed the highest efficiency in reducing biofilm formation (average EBF = 14.08) among the ten tested Ps. aeruginosa strains, followed by AZM > CLR. > SP > E > T (average EBF = 58.8).Regarding the ability of macrolides to eradicate the already formed biofilm, the effect of lethal concentrations; 2 x MIC, of the different macrolides on the ready formed biofilm by the selected Ps. aeruginosa strains was determined. The different isolates had variable response to the procedure of biofilm eradication. However, the biofilm formed by the resistant isolates P2 subsequent with P10 was highly susceptible to the procedure employed, as the average log decrease in the number of cells was 2.16 and 2.12 correspondingly. The remaining strains were more or less similarly affected by macrolides; the resultant averages ranged between 1 and 1.8 log decrease in the number of cells. Clarithromycin showed high potency in removing the ready formed biofilm (average EBE = 3.0) among the ten tested Ps. aeruginosa strains, followed by SP > AZM > E > ROX > T (average EBE = 40.9). From these results we can deduce that the use of macrolides to treat infections caused by Ps. aeruginosa is highly recommended even if they are macrolide resistant. This is based on the ability of macrolides to ameliorate disease conditions and help in the progress of treatment via their effect on biofilm formation and eradication.

4/46 SENSITIVITY OF THREE METHODS FOR DNA ISOLATION FROM EGYPTIAN NEGLECTED PLANTS EVALUATED WITH RAPD TEST

Mohamed A. Attia - Ali M. El-Refy and Fawzy Ali El-Feky

Biotechnology Department, Faculty of Agriculture, Al-Azhar University,

Cairo, Egypt.

DNA extraction is a routine step in many molecular biology studies. A variety of methods have been used to isolate DNA molecules from plants, and many commercial kits are available. Isolation of high quality DNA from Egyptian neglected Plants more difficult than other cultivated plants. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during the extraction. Three methods were evaluated for isolation of high yield and purified DNA from Egyptian neglected plants. We used CTAB (Cetyltrimethyl ammonium-bromide) as manual method and two different kits, the Ferments and QIAGEN as different methods to isolate DNA from Citrullus colocynthis, Juncus rigidus, Phragmites mauritianus and Alhagi maurorum. The results show that kits are more effective for DNA isolation with high purity enabled us to have good results from RAPD. In order to setup a DNA extraction protocol very sensitive and specific for neglected plants.
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