full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 5 (3) 1282-1297 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
1283<br />
Micromonospora <strong>and</strong> Actinoplanes (4).<br />
Although C<strong>and</strong>ida spp. occur among normal<br />
body flora in mouth <strong>and</strong> gastrointestinal tract,<br />
they can be responsible for many types <strong>of</strong><br />
diseases in both humans <strong>and</strong> animals ranging<br />
from superficial mucocutaneous illness to deep<br />
life-threatening invasive, systemic diseases that<br />
can occur in different organs thus, causing<br />
different signs <strong>and</strong> symptoms. There are more<br />
than 20 species <strong>of</strong> C<strong>and</strong>ida, <strong>of</strong> which C<strong>and</strong>ida<br />
albicans is the most common. Yet, all C<strong>and</strong>ida<br />
spp. cause the same spectrum <strong>of</strong> diseases<br />
however, differences in disease severity <strong>and</strong><br />
susceptibility to antifungal agents were reported.<br />
Among the increase in the incidence <strong>of</strong><br />
nosocomial fungal infections, those caused by<br />
C<strong>and</strong>ida albicans, has increased proportionally.<br />
This problem appears to be a global one; in large,<br />
university-affiliated hospitals (5).<br />
Therefore, new sources <strong>and</strong> strategies are<br />
required to find new broad-spectrum antifungal<br />
molecules to combat these pathogens. For this<br />
reason, intensive program searching for new<br />
antibiotics is running worldwide mainly by<br />
screening for members <strong>of</strong> the clade<br />
actinomycetes as being a rich, unrivaled source<br />
<strong>of</strong> antibiotics.<br />
Impressed by these facts, our work aims to<br />
investigate the potentialities <strong>of</strong> some locally<br />
isolated actinomycetes to produce bioactive<br />
compounds acting against C<strong>and</strong>ida albicans<br />
pathogen. The optimal fermentation conditions<br />
for bioactive compound production by the most<br />
active actinomycetes isolate have been<br />
investigated. In addition, trials to identify the<br />
active substances were reported.<br />
Materials <strong>and</strong> Methods<br />
Isolation <strong>of</strong> actinomycetes : Stone samples were<br />
crushed to a fine powder. Stone powder was<br />
dispersed in sterile 1/4 strength Ringer’s solution<br />
supplemented with 0.001 % Tween 80 <strong>and</strong><br />
El-Refai et al<br />
shaken vigorously for up to one hour (6).<br />
Suspensions were then serially diluted in<br />
phosphate buffer. Spread plate method was<br />
applied for actinomycetes isolation; 0.1 ml <strong>of</strong><br />
the appropriate dilution was spread evenly over<br />
the corresponding overnight dried medium<br />
plates. Five different isolation media known to<br />
support the growth <strong>of</strong> actinomycetes were used<br />
namely; starch casein medium (7), arginine<br />
glycerol salts medium (9), starch nitrate medium,<br />
M3 medium (9) <strong>and</strong> ISP medium 5 (Glycerol-<br />
Asparagine Agar) (10).<br />
After incubation, representatives <strong>of</strong> each<br />
colony form were picked <strong>of</strong>f <strong>and</strong> streaked to<br />
obtain a pure culture. They were finally stored<br />
on starch casein slopes at 4 °C, <strong>and</strong> as spore<br />
suspensions in 20 % glycerol at 20 °C (11).<br />
Target organisms : The isolated strain activities<br />
were tested against: Escherichia coli ATCC<br />
25922, Pseudomonas aeruginosa ATCC 27953<br />
<strong>and</strong> Alcaligenes faecalis NRRL B-170 as<br />
examples for Gram negative pathogens,<br />
Staphylococcus aureus ATCC 29213, Bacillus<br />
subtilis NRRL B-4219 <strong>and</strong> Micrococcus luteus<br />
NRRL B-287 as examples for Gram positive<br />
pathogens, Aspergillus niger NRRL 599,<br />
Aspergillus flavus NRRL 1957 <strong>and</strong> Trichophyton<br />
mentagrophytes ATCC 9533 as examples for<br />
fungal pathogens <strong>and</strong> C<strong>and</strong>ida albicans ATCC<br />
10231, Saccharomyces cerevisiae ATCC 10275<br />
as examples for yeasts.<br />
The challenged fungi were maintained <strong>and</strong><br />
cultured on Dox’s agar medium that has the<br />
following composition (g/l): sucrose, 20; NaNO 3<br />
,<br />
2; MgSO 4<br />
.7H 2<br />
O, 0.5; KCl, 0.5; FeSO 4<br />
.7H 2<br />
O, 0.1;<br />
agar, 20. The pH <strong>of</strong> the medium was adjusted to<br />
7.0-7.2 before sterilization.<br />
The tested yeasts were maintained <strong>and</strong><br />
cultured on malt yeast medium that has the<br />
following composition (g/l): glucose, 4; yeast<br />
extract, 4; malt extract, 20; agar, 20. The pH <strong>of</strong>