Egypt. J. Exp. Biol. (Bot.

), 12(2): 275 288 (2016) Th e Eg yptian Society of Exp erimen tal Biolog y
DOI: 10.5455/eg yjebb.20161221102915
RESEARCH ART ICLE

Dalia A. M. Abdou
Nour a A . El-F ar
Youssria M. S het aia
W ael S. I. Ab ou-Elm agd
Al-Zahr aa A. K aram El -Din

EXPL OR IN G T H E AN T I M I C R OBI AL POT EN T I AL OF L OC AL M AR IN E F U N GI

ABSTRACT:
In s earch f or bioactive c ompounds, 88 f ungal
isolat es w ere c ollect ed from t hree Eg yptian
marine habitats . Crud e extrac ts of 17 m arine
derived fungal is olat es out of t he 88 isol at es
show ed v ariable activit y ag ainst som e human
pathogenic bac terial strai ns ( S almonella
enterica, Prot eus sp., Klebs eilla pneumonia,
Staphyloc occ us aureus and Escherichi a coli ).
The m ost pot ent fungal isol at e that s howed
the highes t antimicrobial activit y
molec ularl y identified as As per gillus
welwetsic hiae. Optimizati on of c ultur e
conditions of A. wel wet sichi ae r evealed t hat
the highest antimi cr obial activit y w as obtai ned
in s haki ng c onditions (150rpm), using
Czapek- dox medium, at pH 9 and incubation
at 25C f or 3 d ays. S almonella ent erica w as
the most s ensitive t est organism regarding the
highes t inhibition z one t hrough all the
optimization tests. E thyl ac et at e extr act of A .
welwetsic hiae w as s ubject ed t o c hemic al
anal ysis and declar ed t hat t he activ e
antibact erial m et abolite is a mi xtur e of acid
(Mono (2-et hyl hexyl) pht halat e) and est er (bis
(2-et hylhexyl) pht hal at e). Ret esti ng the tw o
compounds s eparat el y, they s how ed no
activit y whic h confirmed the s ynergistic
antibact erial eff ect of t he two c ompounds
toget her. Upon testing the c yt ot oxic eff ect of
the active antibac terial m et abolit e (mixtur e of
acid and ester) ag ains t human lung fibroblast
norm al cell (W I-38 c ell line), t he res ults
show ed t hat there w as a ver y w eak inhibitor y
activit y s uggesting the possibility
considering it as a saf e c ompound f or f urther
steps as a pharmac eutical prod uct.
KEY WORDS:
Marine derived f ungi , A ntimicrobial
compounds, As per gillus
CORRESP ONDENCE:
Dalia, A.M. Abdou
Microbiol ogy D epartment , F ac ult y of Science,
Ain-S ham s Univ ersit y, Cairo, Eg ypt.
E-mail: drdaliaali2013@yahoo. com
*Nour a, A. El -Far
* Youssria, M. S het ai a
** W ael S . I. Abou-Elm agd
*Al-Zahraa, A. Kar am El -Din
* Microbiol ogy D epartm ent , Fac ult y of
Science, Ain-S hams U niversit y, C airo,
Egypt.
** Chemistr y D epartm ent , F acult y of Science,
w as Ain-S ham s Univ ersit y, Cairo, Eg ypt.
ARTI CLE CODE: 28.02.16
I NTRODUCTI ON:
Ther e are two maj or t yp es of
biologically import ant environm ents in whic h
the s alt f act or will i nt erac t wit h microbial
populati ons , soil and water (Mansum a et al. ,
2001). The oceans and seas c ov er 70% of the
eart hs sur fac e and poss ess a wide di versit y
of nat ural flor a and f auna. Among the 36
know n living phyl a, 34 ar e f ound in m arine
envir onm ents with more than 0.3 million
know n speci es of fl ora and f auna (Faulkner,
2001).
of
New tr ends in drug discov er y from
natural s ourc es emphasiz e on i nvestiga ti on of
the m arine ecos ystem t o explore num erous
complex and novel c hemic al entities. Thes e
entities ar e t he s ource of new lead f or
treatment of m any diseases s uc h as canc er,
A IDS , i nflamm at or y condition, art hritis,
malaria and large v ariet y of viral, bact erial
and fungal diseases (N azar et al. , 2009).
Bec ause of t he highl y c hemical and physic al
hars h c ondition i n marine environm ent, the
organisms produc e a v ariet y of m olec ules wit h
unique s truct ural feat ures and exhibit v arious
biological ac tivities (Ravik umar et al., 2010).

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276
Marine f ungi are prolific r es ourc es of
natural prod ucts ( Ebel, 2010; J ensen and
Fenic al, 2002). H ow ever , t he potenti al of
marine fungi has onl y been inv estigat ed t o a
limited extent. In t he last few dec ades ,
marine-deriv ed f ungi have b een rec ogniz ed as
one of the m ost rec ent bar el y t apped s ourc es
for new biol ogicall y activ e sec ondar y
metab olites (J ens en and F enic al, 2002)
including antitum or, antibac terial, antiviral,
antifungal, anti -infl ammat or y and enz ym e
inhibitor comp ounds. This is probabl y
because mari ne f ungi hav e been explor ed t o a
lesser ext ent t han t heir terrestrial
count erparts, w hic h hav e been k nown for a
long tim e as a ver y important source of
biologically activ e and ec onomic ally import ant
natural produc ts, suc h as t hose f or us e i n
treatment of hum an diseases as well as other
biotec hnologic al applications (T an and Z ou,
2001; Strobel , 2002).
Aspergillus f ungi hav e rec eived the
most of t he att ention am ong all the marine -
derived f ungi, w hich account ed f or 31% of the
marine fung al origin. The m arine nat ural
products have divers e c hemical struc tur es
mainl y incl uding; pol yk etides , fatt y acids,
sterols and terpenoides, 96% of whic h
displayed bioac tivities s uch as c ytotoxicit y,
antimicrobial activit y, antioxidant and
insec ticidal ac tivit y (Z hao, 2016).
B act erial resistanc e is spreading
throughout t he w orld; especi all y in all healt h
care ass ociated p athogens r evealing the
steadily d ecreasing p ot enti at es of preval ent
antibiotics (G ould, 2008); t hus, necessitating
the discov er y of n ov el compounds,
modification of alread y existing antimicrobial
stock , be it from fungi, ac tinom yc etes or any
natural r esourc es. Bec ause of huge
expenditure on s ynthetic m olec ule wit h
eff ectiv e antimicrobial properti es, nat ural
products ar e still a wort h pr omise. Alt hough,
more suc h organisms for t heir antimicrobial
potential t o solv e t he problem of emerging
strains of r esist ant microorganisms (K aur and
Arora, 2015.
The pot ential of marine fungi t o produc e
a vast array of sec ondar y metabolit es t hat ar e
gaining importance f or t heir biotechnol ogical
as w ell as m edical and pharm ac eutic al
applications has at tract ed our att enti on.
Howev er, the aim of t his st ud y is t o highlight
the occ urrenc e of loc al marine f ungal isol at es
havi ng t he c hemical pot ential of bioactiv e
metab olites with antimicrobial activit y.
M ATERI AL AND M ETHODS:
Marine f ungi are an ecol ogicall y rat her
than physi ologicall y or t axonomicall y defi ned
group of microorganisms (H yde et al ., 1998).
Col l ecti on of w ater sam pl es:
W ater samples w ere c ollect ed fr om
three diff erent sit es i n Eg ypt; R ed Sea (Sharm
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Egypt. J. Exp. Biol. (Bot.), 12(2): 275 288 (2016)
El-Sheikh), Mediterranean S ea (Al exandria)
and Bi tter l akes ( Ismailia). Fr om eac h
locati on, samples w ere coll ect ed in cl ean
sanitiz ed bottl es at a depth of 1. 5 meter using
LaMott e w at er sampler (m odel JT - 1 c ode
1077). Thes e s amples w ere c ollec ted (during
March and April, 2012), tr ans ferred t o the
laborat or y and st ored in refriger ator f or
furt her anal ysis.
I sol ati on of M ari ne Fungi :
Three differ ent cult ure m edia w ere used
for is olati on of marine fungi; Sab our aud
dextros e yeast extr act ag ar medium (Feng et
al., 1990), Cz apek- D ox ag ar medium (Thom
and C hurc h, 1926), and Yeas t extract m edium
(Wickerham, 1951).
Is olation media w ere prepar ed with sea
wat er and supplement ed with chl oramphenic ol
(250 mg/L) as antibact erial agent. T he agar
plates were inoc ulat ed wit h w at er s amples
(0.2 ml) and inc ubat ed f or t hree w eeks. The
agar plat es w ere reg ularl y exami ned t o v erif y
the growing c olonies. Disti nct fungal col oni es
on the is olati on media w ere then transf erred
to new p lates f or f urt her purification. The
single purifi ed col oni es w ere picked up and
subcult ured on slants prep ared from t he sam e
contents of the isol ati on m edia and
maint ained for f urt her inv estigations (S amuel
et al., 2011).
Assessm ent of Anti m i crobi al Activi ty of
M ari ne Fungal I sol ates:
P repar ati on of the aqueo us crud e extr act:
Eac h 50 ml of Cz apek -Dox brot h
medium (pH 6. 5) i n Erlenm eyer flasks ( 250
ml) was inoc ulat ed with one plug of eac h
fungal isol at e and inc ubat ed in s haking
incubator at 150 rpm, at 28C for 7 days
(Zainuddin et al ., 2010). T he f ung al m yc elia
were harves ted by filtr ation thr oug h W hatman
filter pap er N o. 1, the sup ernat ant w as t hen
filtered t hrough two t ypes of Millipore S yri nge
filters (0. 45 m) and (0. 22 m) to g et c ell free
extract (Manim egalai et al. , 2013). The
sterilized c ell free filtr ate was used f or
screening of antibact erial and antif ungal
activities agains t the t es ted bacteri al and
fungal pat hogens.
Antim i crobi al acti vi ty test:
Agar w ell diffusion m et hod w as us ed t o
eval uate t he antimicrobial ac tivit y of the
sterilized cell free f ungal extract s (Mat han et
al., 2013) against som e hum an bacteri al and
fungal pat hogens. T he bac teri al pat hog ens ;
Salm onella ent erica ATCC 25566, Pr oteus sp.
(clinical is olat e), Kl ebs eilla pneum oni a ATCC
10031, St aphyl oc occ us aur eus ss. aureus
ATCC 6538 and E scherichi a coli ATCC 51659
were purc hased from Microbiol ogical
Resources C entr e (Cair o MIRC EN), F acult y of
Agriculture, Ain Shams U niversit y. W hile
fungal pathogens; As per gillus flav us (kf
028197), A spergillus ni ger (kf 358715) and
Aspergillus f umigat us (k f 201647) w ere kindl y
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A bdo u e t a l . , E xp lo r i ng t he A nt i m i c r o b i a l P o t e nt i a l o f Lo c a l M a r i ne F u ng i
supplied by personal c ontac t. Candida
albic ans was obt ained from c ult ure c ollecti on,
Department of Microbiol ogy, F acult y of
scienc e, Ai n-shams U niversit y.
The st erilized f ung al aq ueous extracts
(200 l) were loaded in t he w ells punched in
agar plat es seeded wi th differ ent diluti ons of
spore s uspension (c onc entration eq uival ent t o
standard McF arland 0. 5) of the diff erent
test ed pat hogens. F or bact erial pat hog ens ,
nutrient agar plat es ( APH A, 1917) wer e
seed ed with spor e s uspension (10 8 ) and
incubated at 37C f or 24hrs. Cz apek -Dox agar
plates wer e s eed ed wit h sp ore susp ensi on of
(10 4 ) of t he fungal pat hog ens and inc ubated
for 7 days . Malt extrac t agar plat es wer e
seed ed with spore s uspensi on of yeas t
pathogens (10 6 ) and inc ubated f or 48 hrs.
Fung al and yeas t plat es w ere inc ubat ed at
28C.T he m ean diam et er of inhibition zones
were m eas ured in millimeters and recorded
(Powt hong et al., 2012).
I denti fi cati on of the acti ve m ari ne fungal
i sol ates:
Id entific ati on was d et ermined bas ed on
their macrosc opic and microscopic
morphol ogical c har act eristics using the
univ ersal manual (De H oog et al., 2000). The
identific ation of the mos t pot ent isolate w as
confirmed usi ng t he m olecul ar t echniques.
M ol ecul ar i denti fi cati on of the m ost potent
i sol ate:
Molec ular identification was c arried out
in Sigma C ompany. DN A extr acti on was m ade
by using protoc ol of G ene Plant g enomic DN A
purification kit (T herm o) # K 0 791. Then PCR
was m ade b y using Maxima H ot St art PCR
Mast er Mi x (T herm o) # K 0221: F orward primer
IT S1: (5-TCC GTA G GT G A A CCT GCG G -3)
and revers e IT S 4: ( 5 - TCC TCC GCT T AT
TGA T AT GC-3).
Initial denatur ation at 95C for 10
minut es. Denatur ati on 95C f or 30 sec onds.
Annealing at 55C f or 1 minut e. E xtension at
72C f or 1 minut e. Final ext ensi on at 72C f or
15 minut e. N umber of c ycl es, 35. The PCR
was cl ean up t o the produc t using Gene J ET TM
PCR purificati on Kit (Thermo) # K 0701.
Finall y seq uenci ng to t he PCR product w as
made i n GA TC C ompany using f orward and
revers e primers, AB I 3730xl DN A Seq uencer.
Extr acti on and el uci dati on of the structur e
of the anti m i crobi al m etaboli te from the
m ost potent m ari ne fung al i sol ate:
Extr acti on of anti m i crobi al m etabol i te
(Atal l a et al. , 2008):
Czapek- dox brot h medium ( 100 ml) was
inoc ulat ed with pl ugs of the mos t pot ent
marine f ungal isol at e (No. 9) and incubated i n
a shaki ng inc ubat or (150 rpm) at 25C, for 7
days. Af ter inc ubati on days, fungal m yc elium
was s epar at ed from c ultur e brot h by filtration
through W hatm an N o.1 filt er pap er. F or
extraction of antimicrobial met abolit es, the
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277
filtrate w as treated with eq ual vol umes of four
different organic s olvents namel y hexane,
chlor oform, dichl oromethane and et hyl
acetate. T hen, shaki ng t he mi xt ure vigorousl y
for 10 minut es in a s eparating f unnel. The
miscible mixtur e w as all owed to st and f or 5
minut es and the solv ent p has e c ont aini ng the
fungal antimicrobial m etab olites was c ollec ted
in a fl ask and c onc entrated in a rotat or
evap orat or at 30C. T he obt ained residue w as
dissolved in 2% dimet hy s ulfoxide (D MSO)
and st ored at 4C. Ass essm ent of
antimicrobial activit y was c arried out using
agar well diffusi on m et hod. In nutrient agar
plates wit h 0. 5 McF arland suspension of
Salm onella enteric a, a v olum e of 200 l of
concentrated fungal extrac t diss olved in 2%
DMS O w as l oaded i n w ells punc hed in agar
plates and i ncub at ed at 37C f or 24 hrs.
Similarly, 200 l of 2% D MS O was us ed as a
negative contr ol and Chl oramphenic ol (10 g)
was used as p ositive contr ol.
Extr acti on and chem i cal characteri zati on of
the anti m i crobi al m etaboli te from the m ost
potent m ari ne fung al i sol ate:
P uri fi cati on of the antim i crobi al
m etabol i te:
The activ e c omponents i n the et hyl
acetate crud e extr act wer e separ at ed using
silica gel t hin layer c hrom at ography prec oated
aluminum sheets 60F 25 4 ( Merck). Tw o m obile
phas es w ere used for extracti on; et hyl
acetate: chl oroform (1:1) (v /v) and diet hyl
et her: petrol eum et her ( 1:1) (v/ v). The cr ude
extract w as spot ted at 1. 5cm fr om the b ott om
of t he sheets and all owed t o dry, t hen
develop ed in an ascending order f or an hour.
The produc ed spots w ere located b y t heir
fluor esc enc e on c hrom at ograms under U. V
light and R f (retardation f act or) val ues wer e
determined (At alla et al. , 2011).
El uci dati on of structure of the
anti mi crobi al acti ve m etabol i te vi a
spectro scopi c an al ysi s:
It is difficult t o acc ess t he original
compound of int erest pres ent in nat ural
products whic h is us uall y a c omplex mi xtur e
of s ever al compounds. Thr ee i mport ant
tec hniques w ere undert ak en t o reac h the
struct ure of antimicrobial m et abolite: GC - MS ,
IR and N MR spec trosc op y.
- Fouri er Tran sform I nfrar ed R esonance
spectro scopy (FTI R):
The infr ared sp ectra w ere r ecorded
using pot assi um bromide disks on FT IR
Therm o Electr on Nicol et 7600 (US A) infr ared
spectrom et er at the C entral labor ator y of
Facul t y of sci ence, Ai n s hams universit y.
- Gas ch rom atography m ass sp ectrom etry
(GC-M S)
The m ass spectr a wer e rec orded on
Aglient Tec hnol ogies GC - MS 5977A mass
spectrom et er op erati ng at 70 ev at t he C entral
laborat or y of F ac ult y of s cience, Ai n s hams
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278 Egypt. J. Exp. Biol. (Bot.), 12(2): 275 288 (2016)

- Nucl ear Magnetic Resonance spectroscopy The distribution of marine fungal isolates
(NMR): collected from different sites in Egypt.
The 1 H-N MR spectra w ere m eas ured on Dat a of t his st ud y revealed t hat the yi eld
Varian G emini 400 MHz spectr omet er, wit h of t he c ollect ed s amples w as higher fr om Red
chemic al s hift ( ) express ed in ppm dow nfield sea than other sit es. N adeem et al. (2015)
with t etr amet hylsilane (T MS) as int ernal reported that , R ed s ea w as rec ognized as a
standard, in D MSO-d6 and c oupling c onst ants rich source of microbial diversit y wit h unique
J in Hz. at the m ain c hemical w arfar e metab olites with pharm aceutic al and
laborat ories. medicinal import ance.
Eval uati on of cytotoxi ci ty agai nst WI -38 Assessm ent of anti mi crobi al acti vi ty of the
cel l l i ne: i sol ates:
The purified antimicrobial c ompound All marine- deriv ed fungal isol at es in
was ev aluat ed f or c yt ot oxicit y ag ai nst W I-38 this st udy w ere subjected t o antimicrobial
cell line (human Lung Fibroblast norm al cell). activit y test agai nst a group of pathogenic
The c ell line was obtai ned from VAC SER A bacteria, m old and yeast. As s how n i n table 1,
Tissue C ultur e Unit . T est w as carried out at the crude extr acts of onl y sev enteen (19%)
the R egional Cent er f or Mycol og y & marine- derived f ung al isolates s howed
Biot ec hnolog y, Al -Az har Univ ersit y. activit y ag ainst t he t est ed bact erial
The cells w ere propagat ed in Dulbecc os pathogens. T his data c oi ncides wit h other
modified E agles m edium (D ME M) literat ures (Zhang et al. , 2012 & 2013; Qin et
supplemented with 10% heat -inac tivat ed f et al al., 2015), w here it w as r eport ed t hat 38%
bovine s erum , 1% L-glut amine, HE PE S buff er 59% of t he test extract s from marine f ungi
and 50g/ml gent am ycin. All c ells wer e exhibited antibact erial or antifungal activities .
maint ained at 37C in a humidified Also, Suay et al. (2000) report ed that about
atmospher e with 5% CO2 and wer e 70% of fungal s trains w ere activ e agains t
subcult ured tw o times a w eek. bacteria.
The cells w ere seeded in 96-well plat e Table 1. Assessment of antibacterial activity of the active
marine fungal isolates (17 isolate).
at a c ell concentr ation of 1 10 4 c ells per
well. T est ed extrac ts diss olved i n D MSO wer e Diameter of inhibition zones in mm
added to t he w ells in triplicates wit h Staphylococcus
Isolate Escherichia Klebseilla Salmonella Proteus
concentrations of 0, 15.60, 31. 25, 62. 50, aureus ss.
number coli pneumonia enterica sp.
aureus
12.50, 250, and 500 g/mL for 48 hrs. The
1 - 18 - 25 -
cytot oxic activit y was d et ermined using
2 20 19
viability ass ay. The optical d ensi t y w as
meas ured with t he microplate reader 3 - 17 - - 17
(SunRise, T ECAN, Inc, US A) to det ermine the 4 - - - 30
number of viable c ells and the percent age of 5 - - - - 17
viability w as calc ulated as [ 1-(ODt/ ODc)] x 6 - 25 - - -
100% where ODt is t he m ean optical densit y 7 - - - 25 22
of w ells treat ed with t he tested s ample and 8 - - - 25 18
ODc is the mean optical densit y of untr eated 9 19 - - 24 19
cells. T he r elati on betw een survivi ng c ells and 10 13 - - 20 18
drug concentration is plott ed t o g et the 11 - - - 21 18
survival c urv e aft er treatm ent wit h the 12 - 30 - - -
specified comp ound ( Mosmann, 1983; G omha 13 - 27 - - -
et al., 2015). 14 - 24 - 20 -
Stati sti cal Resul ts: 15 - - - 30 25
16 14 - - 22 17
Statistical anal ysis of experimental dat a
was perf ormed using IB M SP SS 2011; t o 17 - - 18 21 -
detec t t he optimum conditions f or maxim um
Is olates number 9, 10 fr om (R ed s ea)
antibact erial activit y of t he m ost pot ent
and N o. 16 from (Bit ter lak es) s howed ac tivit y
marine- deriv ed fungal is olate. The probabilit y
agains t m ost of t he t est ed bact erial
of error (P val ue) at 0. 05 or less w as
pathogens, where t he diameter of i nhibition
consid ered signific ant, w hile at 0. 01 and
zones ranged from 13mm i n cas e of E . coli t o
0. 001 highl y signific ant.
24 mm i n cas e of S almonella enterica.
Accordingl y, is olat e N o. 9 had b een s elec ted
RESULTS AND DI SCUSSI ON : for f urther inv estigations as being t he mos t
I sol ati on of m ari ne fungi: potent is olate wi th the highest antibact erial
activit y (inhibition zones rang ed from 19 mm
In this study, eighty-eight marine fungal
to 24 mm).
isolates were collected from the three different
isolation sites in Egypt; 42 isolate (48%) from In this st ud y, antibact erial ac tivit y w as
Red Sea, 32 isolate (36%) from Bitter lakes found t o be m ore eff ectiv e t ow ards Gram -
and 14 isolate (16%) from Mediterranean Sea. negative bact eria than Gram -positiv e bac teria.

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A bdo u e t a l . , E xp lo r i ng t he A nt i m i c r o b i a l P o t e nt i a l o f Lo c a l M a r i ne F u ng i 279

Thes e r esult s wer e diff erent fr om t hat of dominant g enus r epresenti ng 33% foll owed b y
Christophers en et al. (1999), H ller et al. Penicillium 13% of t he m arine f ung al isol at es
(2000), and S uay et al. (2000) w her e t hey collect ed in his st udy. Xu et al. (2015) in t heir
reported t hat antibact erial activit y is mor e stud y, rep orted t hat dominant genera of the
common t ow ards Gram -positiv e bac teria t han 105 marine fungi wit h antibac terial and
Gram-negativ e bac teri a. T hese differenc es i n antifungal ac tivit y w ere speci es of t he genus
susc eptibility tow ards antibiotic ally activ e Aspergillus ( 31 s train) and t he genus
sec ondar y m et abolic extracts hav e b een Penicillium (16 s trai n), and considered
repeat ed l y attribut ed t o diff erences in c ell wall Aspergillus as one of t he dominant marine
struct ure of Gr am -positive b act eria c ompared fungi and str ains related to t his genus
to Gram-negative b act eria. The c ell w alls of producing more new antibact erial and
Gram-positive bact eria are l ess c omplex and antifungal comp ounds than any ot her genus .
lack the nat ural si ev e eff ect agains t l arge Also, they rep orted t hat, over 700 c ompounds
molec ules (H awk ey, 1998), whereas th e out er in t ot al wer e purified from 105 fungal strai ns
membrane and t he periplasmic sp ace t hat is that c an produc e antimicrobial c ompounds
present in Gr am -negativ e bact eria is t hought and were i nves tigat ed f or t heir activities .
to provide an additional degree of protec tion Ther e are 285 c ompounds (approximat el y
agains t antibiotics t argeti ng the cell wall 40% of t he tot al) s howed antibac terial and
(Basile et al., 1998). B y t hat f act, i n t his antifungal ac ti vities and 116 (15% of the total)
stud y, t he mari ne- derived fungal isol at es are new antibact erial and antif ungal
from thos e regions in Egypt are of great compounds. As per gillus f ungi have r ec eived
importanc e since t hey ar e mor e ac tive agains t the mos t of att enti on am ong all marine
Gram-negativ e bac teria. derived f ungi, w hich account ed f or 31% of the
Identification of the active marine fungal isolates: marine fungal origin (Zhao et al., 2016).
The s event een marine-derived fungal As show n in t he r esult s of table 2,
isolat es w hich gav e v ariable activit y agains t members of As pergillus ni ger (group) wer e
the t est ed bact erial pat hog ens wer e identified represent ed by 12 ( 82%) out of 14 m arine
traditionall y by t heir macr osc opic and fungal isol at es bel onging t o As per gillus
microscopic c har act eristics (Table 2). W hile genus. As per gillus niger as an example of one
the identific ation of t he m ost pot ent f ungal of the species r elat ed t o As per gilllus niger
isolat e (N o. 9) was c onfirmed using the (group) is one of th e bes t pharm ac eutic al
molec ular t ec hniques. In the c ur rent st udy, friendl y organism t hat produc es a lot of
out of t he 17 active is olat es 14 (82%) industriall y important enz ym es as w ell as
belong ed to genus As per gillus, tw o some other products. Rel at ed t o antimicrobial
Penicillium species ( 11%) and one activit y, it s hows high potenc y i n producing
Cladosporium species (Table 2) . In antimicrobial c ompounds such as tens yuic
acc ordanc e to our res ults , D as et al. (2009) acid, nigerazi ne B, t ensidol A and oc hratoxi n
reported t hat , As per gillus was the mos t (Nielsen et al. , 2009).
Table 2. List of the active marine fungal isolates and their antibacterial activity
Isolate number Identification Isolation site Activ e against
1 Penecillium waksmanii Meditteranean S ea K, Sa
2 Aspergillus niger (gro up) Meditteranean S ea Sa, P
3 Aspergillus niger (gro up) Meditteranean S ea K, P
4 Aspergillus flavus (gro up) Meditteranean S ea Sa
5 Aspergillus niger (gro up) Meditteranean S ea P
6 Aspergillus niger (gro up) Red Sea K
7 Aspergillus niger (gro up) Red Sea Sa, P
8 Aspergillus niger (gro up) Red Sea Sa, p
9 Aspergillus niger (gro up) Red Sea E, Sa, P
10 Aspergillus niger (gro up) Red Sea E, Sa, P
11 Aspergillus niger (gro up) Red Sea Sa, P
12 Aspergillus flavus (gro up) Bitter Lakes K
13 Cladosporium sphaerosperm um Bitter Lakes K
14 Aspergillus niger (gro up) Bitter Lakes K, Sa
15 Penecillium waksmanii Bitter Lakes Sa, P
16 Aspergillus niger (gro up) Bitter Lakes E, Sa, P
17 Aspergillus niger (gro up) Bitter Lakes St, S a
Note: K for Klebseilla pneumonia, Sa for Salmonella enterica, E for Escherichia coli, P for Proteus sp. and St for
Staphylococcus aureus ss. aureus.

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280 Egypt. J. Exp. Biol. (Bot.), 12(2): 275 288 (2016)

M ol ecul ar i denti fi cati on of the m ost potent

i sol ate (No.9):
A sequence database search using the BLAST
search program analysis of the National Center for
Biotechnology Information (NCBI, http://www. ncbi.
nlm.nih.gov/BLAST/) against various sequences was
used to identify the phylogenetic similarities among
the Aspergillus niger (group) isolates and the
published DNA sequences in GenBank. The BLAST
search demonstrated that this isolate was closely
related to As pergillus welwit schi ae with sequence
similarity of more than 98% to the ITS1-5.8S-ITS2
regions of the rRNA genes using ITS1 and ITS4
primers and this was sufficient to indicate that this
isolate belongs to the same species (As per gillus
welwi tschi ae) (Figs 1 & 2).
Fig. 1. Amplified PCR product of the most potent isolate

Fig. 2. The phylogenetic tree of Aspergillus twelwitschiae (98% similarity).

Opti mi zati on of cul ture condi ti ons of while all ot her cultiv ati on param et ers
Aspergillu s welwit sch iae: remained unc hanged (B hat tac har yya and Jha,
2011; R amos and Said, 2011). In all t ests ,
The yi eld of bioactive compounds can
antibact erial ac tivit y w as m eas ured as
sometimes be s ubst antiall y incr eas ed by the
diameter of inhibition zone in mm .
optimization of physic al (temper at ure, s alinit y,
pH and light) and chemical f act ors (media Effect of m edi um com posi ti on on
components, prec ursors, and inhibitors) f or anti bacteri al acti vi ty of Asperg illu s
the growt h of microbes (Mi ao et al ., 2006; wel witschi ae:
Ritchie et al., 2009; J ain and P undir, 2011; Sel ection of a s uitable m edium for
Sudark odi et al. , 2012). antimicrobial produc tion is an important st ep;
Antimicrobial activit y was first t est ed in suc h a m edium w as a prer equisite f or f urther
both st atic and shak en c onditions ( 150 rpm). studies (S hukla et al., 2014). Thr ee diff erent
The obt ained r esults indic at ed t hat , media namel y; Czap ek -Dox brot h, Gluc os e
antimicrobial activit y of shak en c ultur e w as Pept one Yeast extr act brot h and P ot at o
higher than st atic one. For t his reas on, Dextrose brot h ( Murray et al. , 2007) wer e
experiments i n t his st ud y w ere c arried out i n test ed. The antimicrobial activit y w as
shaken c ondition at 150 rpm. Duplicates of ass essed aft er 7 days of inc ubation at 28C.
Erlenm eyer fl asks (250 ml) cont aini ng 50 ml In this st ud y, the antibac terial activit y
broth media us ed to d et erm ine the of Aspergillus wel witsc hiae w as recorded
physiologic al and physic al c onditi ons t hat onl y on Cz apek-D ox broth m edium and the
would aff ect t he antibact erial activit y of highes t i nhibition z one (30 mm) w as
Aspergillus wel witsc hiae. Par amet ers of obtai ned ag ainst Salm onella enterica (T able
cultur e conditions wer e changed one at a tim e 3). This res ult indicated t he s uit ability of
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A bdo u e t a l . , E xp lo r i ng t he A nt i m i c r o b i a l P o t e nt i a l o f Lo c a l M a r i ne F u ng i
Czapek-Dox brot h m edium in the
achi ev ement of the highest antibact erial
activit y of A. welwit schi ae. W hile, other
studies r eported t hat potat o dextros e brot h
was t he b est m edium f or the gro wt h and
enhanc ed producti on of sec ondar y
metab olites from As per gillus t erreus
(Mathan et al., 2013). K aren et al. (2013)
test ed the import ance of differ ent m edia
typ es, i ncluding si x liquid and fiv e s olid
media on the sec ondar y met abolit e
production of t hr ee fungal strai ns in t heir
drug -discovery screeni ng process. T hey
found a s urprising result that t he am ount of
extract produc ed in liquid cultur es was of ten
so drastic all y sm aller t han thos e produced
in solid m edia c ultur es. Als o, produc tion
varied widel y betw een the t yp es of the
test ed media, but gener ally, rice appeared
to be consist entl y produci ng high q uantiti es
of t he activ e metabolit es.
Effect of tem per atur e on the anti bacteri al
acti vi ty of Asperg illu s welwit sch iae:
In t his s tud y, thr ee set s of fl as ks
contai ning Cz apek -dox brot h (pH 6. 5, i n
shaking condition 150 rpm, f or 7 d ays) wer e
incubated at t hree diff erent temper at ures (25,
30, and 35C). O ne-w ay ANO VA of normal
data was c arried out and as shown i n table 3,
there w as a significanc e diff erence b etw een
diameters of i nhibition zones by As per gillus
welwi tschi ae agains t Salm onella ent erica, E .
coli and Prot eus s p. and incubation
temperatur es at 25C, 30C, and 35C. B y 28C for 7 days at thr ee diff erent rp m
Pos t- hoc test, it w as prov ed t hat t emper atur e
25C was t he optimum t emperatur e w ith 0. 006
level of significanc e (P = 0.006).
Bhatt ac har yya and J ha (2011) r eported
that inc ubation t emperat ure r anging fr om
25C t o 35C to be optimum for antimicrobial
activit y of As pergillus s trai n. Suj a et al.
(2013) f ound t hat inc ubati on t emper atur e
(25C) enhanc ed the antimicrobial c ompound
production of As per gillus t erreus , als o K al yani
et al. ( 2016) rec orded 30C as t he bes t
incubation temp erat ure f or Aspergill us niger
(MTTC- 961). T he r ecorded r es ults agree wit h
our r esult s.
Effect of pH on th e anti bacteri al acti vi ty of
Aspergillu s welwit sch iae:
The initi al pH of Cz apek -Dox brot h
media w as adjust ed at t hree diff erent pH
values (pH 5, pH7 and pH 9) usi ng 1N NaOH
and 1N HC L. Flasks w ere inc ubated under
shaking c onditions ( 150rpm) for 7 days at
28C (Smit ha and R osamm a, 2014). O ne- w ay
ANO VA of normal data w as carried out and as
show n i n t able 3, that there was a significanc e
difference betw een diameters of i nhibition
zones b y As per gillus wel witsc hiae agains t
Salm onella enteric a (p = 0.001) and Prot eus
sp. (p = 0. 003) and pH v alues ( 5, 7, and 9)
and b y Pos t-hoc t est, it w as prov ed that pH 9
was t he optim um pH val ue ag ainst bot h
Salm onella enteric a and Prot eus s p. T her e
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281
was no signific ance differ enc e b etw een
diameters of i nhibition zones by As per gillus
welwi tschi ae ag ains t E . c oli. (p = 0.270) under
different pH v alue t est ed.
The pH of a c ult ure medium is us uall y
not const ant thr oughout f ermentation and the
changes that occ ur ar e highl y dep end ent on
compositi on of t he m edium ( Shukla et al. ,
2014). T hongwai and K unopak arn (2007)
point ed out that mos t of microorganisms can
synt hesis antimicrobial comp ounds at pH
ranging from 5. 5 to 8. 5. J ain and P undir
(2011) report ed t hat maxim um bioactiv e
metab olite prod uction i n t heir s tud y w as at pH
6. 0, also Mat han et al. (2013) r ecorded pH 5. 5
was optimum f or As pergillus terreus , thes e
studies sugges t t he acidophilic c har act eristics
of the active is olat es. Likewis e, optim um pH
for the growt h and sec ondar y met abolit e
production of F usarium s olani w as f ound t o be
6. 0 by Mer lin et al. (2013). O n t he c ontrar y, i n
the current st udy, t he highest antibact erial
activit y w as observ ed when A. wel witsc hiae
was grow n at pH 9 and inhibition z one
reac hed 32mm ag ains t Salmonella enteric a
(Table 3). Inhibition z ones w ere signific antl y
less w hen t he f ung us w as grown at neutral
and acidic pH.
Effect of agi tati on speed on th e
anti bacteri al acti vi ty of Asperg illu s
wel witschi ae:
In t his st ud y, i noc ulated fl asks wit h
Czapek-Dox brot h m edium w ere inc ubat ed at
(100,150, and 200).
For agit ation speed; one- way AN OV A of
norm al dat a was carried out and as s hown i n
table 3, there w as a significanc e differenc e
between diameters of inhibition zones b y
Aspergillus wel witsc hiae agai nst E . c oli (p =
0. 029) and Prot eus sp. (p = 0. 045) and
different agitation sp eeds (100, 150, and 200)
and by Pos t- hoc t est , it w as proved that 150
rpm was the optimum agit ation speed agains t
both E. c oli and Prot eus s p. N o signific ant
difference w as obs erved b etween diam et ers
of i nhibition z ones by As pergillus wel witsc hiae
agains t Salm onella ent erica (p = 0. 100) under
different agitati on speeds.
It has b een r ep ort ed pr evi ousl y t hat a
str ai n of As per gill us f umi gat us pr od uc ed
bioac tiv e m etab oli t es on gr owt h m edi um ,
mai ze and c omm er ci al anim al feed wi t h
rot ar y s hak er at 150 rp m (W enehed et al. ,
2003; Li u et al ., 2004). T his r es ul t al s o
corr ob or at ed wi t h t hos e m enti oned b y A t all a
et al. ( 2008) w ho i nc ubat ed the m ari ne
fung us nam ed V ari c os pori na r amulos a on a
rot ar y shak er at 150 rpm f or bi ol ogic all y
ac tiv e c omp ounds pr od uc ti on. Di ff er ent fr om
our r esults and the ot her m enti oned s t udi es ,
K aur and Ar or a ( 2015) rep or t ed hig hes t
anti micr obi al ac tivi t y at 200 rpm and
rem ai ned s am e till 250 rpm .
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282 Egypt. J. Exp. Biol. (Bot.), 12(2): 275 288 (2016)

Effect of i ncub ati on peri ods on th e
anti bacteri al acti vi t y of Asperg illu s
wel witschi ae:
Time duration req uired for growth and
metab olite producti on b y diff erent fungi vari es
significantl y. T hus , proper det ermination of
specific i ncub ation r equirem ent is of high
importanc e f or maxim um harv esting of the
metab olite (Alberts, 1990). Inocul at ed flas ks
of Cz apek-D ox broth medium w ere inc ubated
at 28C f or different i nc ubation peri ods (3
days, 5 d ays, 7 days, 9 days, 11 days , and 13
days) in s haking c onditions at 150 rpm.
Antibact erial activit y w as recorded ev er y tw o
days f or eac h s et of flask s.
One- w ay ANO VA of normal dat a w as
carried out and as shown in t able 3, t her e w as
a signific ant diff erence betw een diam et ers of
inhibition zones b y As pergillus wel witsc hiae
agains t E. c oli, Salmonella enteric a and
Prot eus s p. and diff erent inc ubati on periods
and b y Pos t- hoc t est , it w as prov ed that 3 -
days inc ubati on period w as t he optimum
incubation period with 0. 001 level of
significance ( P = 0.001).
As s hown in t able 3, t he produc tion of
antibact erial metabolit e v aried signific antl y
with i nc ubati on days . Maxim um antibact erial
Table 3. Effect of some factors on the antibacterial activity of the crude extract Aspergillus welwitschiae
activit y w as r ecorded aft er t hree days of
incubation w her e i nhibition zones reac hed 39
mm, 37mm, and 21 mm agai nst eac h of E .
coli, S almonell a ent erica, and Prot eus sp. ,
respectiv el y. A ntibac terial activit y decr eased
graduall y b y incr easing t he inc ubati on period
and no activit y was observed by t he 13t h d ay
of the i ncub ation p eriod. K al yani et al. (2016)
reported m aximum antibac terial met abolit e
production by Aspergillus niger when grow n
for 144 h (6 days). K aur and Arora (2015)
reported the maxim um antibact erial ac tivit y on
5 th d ay of incubation of A spergillus t erreus
which r emai ned m ore or l ess st able till 9 th day
and t hen declined.
The c ult ure st arts goi ng in decline
phas e whic h res ults in red uced bioactivi t y and
that m ay b e due to accum ulation of som e
inhibitor y/ toxic residues ag ainst s ecreted
bioactiv e compounds (Singh et al., 2014). The
time c ourse f or antimicrobial ag ent produc tion
differs ac cording t o t he strai n and c ultivation
conditions . For inst ance, the maxim um
antimicrobial agent produc tion was ac hieved
aft er 5 days of i ncub ation of Penicillium
corylophil um (Silva et al., 2004) and 4 days of
incubation of Cl ados porium sp . ( Miao and
Qian, 2005).

Tested Bacterial Pathogens

Tested factors
Conditions Salmonella enterica Escherichia coli Proteus sp.

CDB 27.50* 2.8** 24.25 2.2 24.50 0.5

1- Medium Composition GPY 0 0 0

PDB 0 0 0

25C 39.75 4.6 29.75 0.9 24.25 1.5

2- Temperature 30C 25.25 2 23.00 2.4 19.00 0.8

35C 0 0 0

pH 5 23.25 0.957 20.25 1.258 19.50 0.577

3- pH pH 7 24.25 2.217 21.50 0.577 24.25 1.258

pH 9 32.25 2.062 23.75 4.787 21.50 1.915

100 rpm 24.50 8.103 19.75 2.217 18.75 0.957

4- Agitation speed 150 rpm 35.00 0.816 23.25 3.096 26.25 1.258

200 rpm 28.25 6.850 21 2.708 23.75 3.775

3 days 36.50 0.577 39.00 0.816 21.00 0.816

5 days 19.25 0.957 20.00 0.816 18.25 1.708

7 days 29.25 0.957 19.50 1.291 18.00 0.816

5- Incubation days
9 days 22.50 0.577 20.00 0.00 20.50 0.577

11 days 18.50 0.577 0 0

13 days 0 0 0

* Results are given as mean value of diameters of inhibition zones measured in mm,
** standard deviation.

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A bdo u e t a l . , E xp lo r i ng t he A nt i m i c r o b i a l P o t e nt i a l o f Lo c a l M a r i ne F u ng i 283

Extr acti on and chem i cal characteri zati on of metab olite giving 29 mm diam et er z one of
the anti m i crobi al m etabol i te from inhibition agains t Salm onella ent erica upon
Aspergillu s welwit sch iae: antimicrobial ac tivit y t est (Fig. 3). N egativ e
control (2%D MSO) s how ed no antibact erial
Extr acti on of anti m i crobi al m etabol ite:
activit y and positiv e control (chl oramphenic ol
Organic s olvents also play an important 10 g ) s howed 57 mm diam et er z one of
role in extracti on of bioactive nat ural produc ts inhibition. T he s ame res ults from t he st udy of
in t he f orm of crud e c ompounds from brot h Lu et al . (2000) and Sun et al. (2011) hav e
media (G out am et al. , 2014). Am ong four used et hyl ac et at e as a solv ent for extracti on,
test ed organic solv ents for extracti on of while J ain and Pundir (2011) f ound t hat
antibact erial m et abolites fr om As per gillus chlor oform w as the bes t solv ent for extrac ting
welwi tschi ae , et hyl acetate was t he onl y Aspergillus metabolit es.
solvent w hich extr act ed antibact erial

Fig. 3. Antibacterial activity of ethyl acetate crude extract

of Aspergillus welwitschiae against Salmonella
enterica
Chem i cal characteri zati on of extr act ed exami ned under U .V light for p urificati on, the
anti mi crobi al m etabol ite from Asperg illu s result s how ed that t he et hyl acet at e c ont ai ned
wel witschi ae. two fl uor esc ent spot s under U .V light ; one
band trav elled thr oug h silica gel sheet wit h an
- Purification of the antimicrobial metabolite:
R F v alue of 0.82 and t he ot her rem ained at
W hen the brow n col ored cr ude the bas eline (Fig. 4).
metab olite extract ed b y et hyl ac et at e w as
spott ed on silica gel TLC s heets and

1stspot

2ndspot

Fig. 4. TLC sheet with dark fluorescent spots produced from extracted antibacterial metabolite

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284 Egypt. J. Exp. Biol. (Bot.), 12(2): 275 288 (2016)

- Structure elucidation of the antimicrobial carboxylic acid, peaks at 3020, 2929, and
active metabolite via spectroscopic analysis 2855 cm - 1 charact eristic f or sp 2 and sp 3 CH
and p eak s c haract eristic for carbonyl groups
To identif y t he extr act ed activ e
at 1741, 1727 and 1645 cm - 1 (Fig. 5). The
compounds i n the separ at ed organic layer,
abov e dat a indicat ed that t here is a mi xt ure of
the FT IR sp ectrum of t he extr act ed met abolit e
acid and es ter.
show ed the appear anc e of broad peak at
(3500 - 3000 cm - 1 ) charac teristic f or OH of

Fig. 5. Infrared spectrum of the purified antibacterial metabolite from Aspergillus welwitschiae
To separ at e the acid, the mixture w as
dissolved in s odium carbonate s olution (50%)
follow ed b y extr acti on b y et hyl ac et at e. The
organic l ayer gave a pur e solid whic h w as
separated and identi fied b y sp ectros copic
data as (bis-2-(ethyl hexyl) phthalate). T he IR
spectrum (Fig. 6) show ed peaks at 2955,
2929, 2852 cm - 1 c har act eristic f or sp3 CH and
peaks c haract eristic f or c arbonyl est er at
1731 cm - 1 . T he assigned str uct ures wer e
supported b y 1 H-N MR and mass spec tra. The
1
H-NMR sp ectra rev eal ed t he exis tenc e of
triplet peak c orresponding t o 4 CH 3 groups at
0.81 - 0.84 ppm, a m ultiplet signals f or
8CH 2 at 1.19-1. 70 ppm, a multiplet signals
for 2CH at 3.70 - 3. 75 ppm , a m ultiplet
signals f or 2 -OCH 2 at 4. 01 - 4.12 ppm and
multiplet signals f or aromatic prot ons at 7.65
- 7. 69 ppm (Fig. 7). The MS spec trum s howed
the correc t mol ecul ar ion peak at 391
corresponding t o M+ 1, beside s om e of
abund ant peaks.

Fig. 6. Infrared spectrum of the ester portion (bis-2-(ethylhexyl) phthalate) of the antibacterial metabolite from Aspergillus
welwitschiae

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A bdo u e t a l . , E xp lo r i ng t he A nt i m i c r o b i a l P o t e nt i a l o f Lo c a l M a r i ne F u ng i 285

Fig. 7. The 1H-NMR spectra of the ester portion (bis-2-(ethylhexyl) phthalate) of the antibacterial metabolite from
Aspergillus welwitschiae.
The c hemic al c har act erizati on of the the mol ecul ar form ula C 16 H 22 O 4 ; mol ec ular
separated acid aft er acidificati on of the weight 279 corresponding to M+ 1, b eside
aqueous layer was det ermined based on some of abundant peaks at m/z(%): 279(12),
elem ent al anal ysis and t he MS spectr al dat a 239(10), 167(30), 149( 65), 97(57), 85(57 ),
(Fig. 8) as Mono (2-ethyl hexyl) pht hal at e wit h 71( 80), and 57(100).

Fig. 8. Mass spectrum of the acid portion (Mono (2-ethylhexyl) phthalate) of the antibacterial metabolite from Aspergillus
welwitschiae.
The chemical characterization of active
fraction extracted from the marine fungus
Phoma h erbarum VB7 was determined based
on the GC- MS and spectral data as Mono
(2ethylhexyl) phthalate (Bhimba et al., 2012).
Atalla et al. (2011) in his study on marine -
derived fungus Penicillium brevicompactum
have suggested that the isolated compounds
which had an antimicrobial effect may be (di
2- ethyl hexyl phthalate) and fungisterol or
one of its isomers. These extracted compounds
from both studies resemble the c ompounds
extracted from the marine- derived fungus
Aspergillus welwitschiae in this study.
Upon testing the antibacterial activity of
each of bis-2-(ethylhexyl) phthalate and (Mono
(2-ethylhexyl) phthalate) separately by agar
good diffusion method against Salmonella
enterica, they didnt exert any antibacterial
activity against the test pathogen unlike testing
them in mixture (acid and ester together) which
was active against Salmonella enterica
confirming the synergistic antibacterial effect
of the two components together.

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286
Evaluation of cytotoxicity against WI-38 cell line:
Upon investigating cytotoxicity of extracted
antibacterial metabolite (mixture of acid and
ester) towards WI-38 cell line (human lung
fibroblast normal cells), it was shown from the
Fig. 9. Relation between viable cells (%) and drug concentration (g/ml) extracted from A. welwitschiae
CONCLUSI ON:
This c urrent st ud y s uggests As pergillus
welwi tschi ae as a potenti al c andidat e off ering
a bett er sc ope f or the produc tion, purification
and is olati on of broad spec trum antibact erial
compound (especiall y agai nst Gram negativ e
bacteria). Thes e findings i n addition to thos e
rep ort ed f or ot her ac tiv e is ol at es i n t hi s
st ud y ( 16 i solat e) m ay f aci lit at e t he s c al e up
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obtained data (Fig. 9) that there was a very weak
inhibitory activity against human Lung Fibrob last
normal cells detected under these experimental
conditions suggesting the possibility of
considering it as a safe compound for further
steps as a pharmaceutical product.
and f urt her purific ation to asc ert ain the
compound r esponsible f or antimicrobial
activit y and pharm aceutic al applications.
ACKNOWLEDGM ENTS:
Deep thanks and appreciations to Dr. Abdel
Haleem Saad (Professor at Zoology Department)
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and Dr. Iman El-Kholy for her kind supply with
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**
* * *
*

*

**
Salmonella enterica .
(
. )
Aspergillus welwitschiae
.
Mono (2 ethylhexyl ) phthalate (Salmonella
bis (2-ethylhexyl) phthalate enterica, Proteus sp., Klebseilla pneumonia,
Staphylococcus aureus and Escherichia
. coli ).
Aspergillus
) ( WI-38 . welwetsichiae



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