Synergistic Effect of Antibiotics and Plant Extract to Control Clinical... Implicated in Urinary Tract Infections

Journal of A pplied Sciences Res earch, 5(10): 1298-1306, 2009
© 2009, INSInet Publication
Synergistic Effect of Antibiotics and Plant Extract to Control Clinical Bacterial Isolates
Implicated in Urinary Tract Infections
1
Ghaly, M.F., 2Shalaby, M.A., 3Shash, S.M.S., 3Shehata, M.N. and 3Ayad, A.A.
1
Botany Department, Faculty of Science, Zagazig University, Egypt.
Immunology & Microbiology Department, Faculty of Medicine, Zagazig University, Egypt
3
Botany Department, Faculty of Science, Benha University, Egypt.
2
Abs tract: A total 100 u rin e s a mples were collected from patients examinated for urinary tract infection
in Zagazig Univers ity hos pitals , Urology Department, Egypt. A pos it ive bacterial growth on CLED,
M acConkey's agar and nutrient agar was detected. E. coli was the mos t pre d o min a n t p a t hogen caus ing
urinary tract infection in pos itive s amples (40%) followed by Proteus mirabilis, Klebsiella pneumoniae,
Staphylococcus saprophyticus and Pseudomonas aeruginosa were pres ent percentage 22, 15, 13 and 10%
res pectively. A ntibiotic s ens itivity tes t for Gram-pos itive and negative bacte ria s howed that the antibiotic
ofloxacin is more effective agains t clinic a l b acterial is olates (58%) followed by amikacin (54%),
chloramphenicol (52%), norfloxa c in (51%) and azithromycin (48%). The effect of medicinal plant extracts
agains t highly res is tant b a c t e ria l is olates s howed that, the clove, ros emary, peppermint, hibis icus , thyme
and cinnamon s howed s trong inhibitory action agains t tes ted is olates . The combination between ofloxacin
and amikacin with clove plant extract s howed that the clearly s ynergis tic effect was obtained agains t tes ted
clinical bacterial is olates . The protein band of P. aeruginosa, P. mirabilis and K . p n e u m o n i ae of normal
growth (con t ro l) a nd under s tres s of combined of ofloxacin and clove extract s howed new protein bands
appeared as res is tant protein to treatment s tres s . The active pure clove s ubs tance s howed antibacterial
activity was identified by 1 HNM R and IR as eugenol s tructure.
Key words : A ntibiotics , plant extract clinical bacteria urinary tract Infections
INTRODUCTION
Urinary tract infection (U T I) remains the mos t
common reas on for outpatients t o s eek medical care
and for inpatients t o d evelop nos ocomial infections .
Nos ocomial UTIs account for up to 40% of all
hos pital-acquired infe ctions . The as s ociated morbidity
and mortality are the major d rain on hos pital
re s ources [3 0 ]. Females are however believed to be mo re
affected than males except at the extremes of life; this
is a res ult of s horter and wider urethra [8] . T h e mos t
common caus e of UTI is Gram-negative bacteria that
be long to the family Enterobacteriaceae. M embers of
th is family include Escherichia coli, Klebsiella,
Enterobacter, and P r oteus. A ls o the Gram-pos itive
Sta phylococcus saprophyticus plays a role in the
bacterial panorama, es pecially among young women. E.
coli dominates as caus ative agent in all patient groups .
In un complicated UTIs , 80-90% are caus ed by E.
coli [2 0 ]. In complicated U TIs , E. coli is les s prominent
but s till the major caus ative agent [2 5 ]. Conventional
drugs us ually provide effective ant ibiotic therapy for
bacterial infections but there is an increas ing problem
of antibiotic res is tance and continuing need for n ew
s olutions [1 9 ]. The etiology of UTI and the an t ibiotic
res is tance of uropathogens have been changing over the
pas t years , b o t h in community and nos ocomial
infection [2 4 ]. The us e of plant extra c t t o treat infections
is a n old practice in a large part of the world ,
es pecially in develop ing countries , where there is
dependence on t raditional medicine for a variety of
dis eas es [1 1 ]. Numerous res earches s howed that the
es s ential oils and plant extracts have potential in
me d ic a l p ro c e d u re s a n d a p p lications in t h e
pharmaceutica l, cos metic and food indus try [1 3 ]. Hers ch
et al.[1 4 ] found that Cinnamomum verum, Origanum
vulgare and Thymus vulgari s extracts , s howed the
highes t and broades t antibacterial activities agains t 189
Gram-negativ e and 135 Gram-pos itive pathogenic
bacteria is olated from p ediatric patients s howed
res is tance to s elected antibiotics . Syzygium aromaticum
have many th e ra p eutic us es ; they relieve pain, control
naus ea and vomiting improve diges tion, protect agains t
internal p a ras ities and act as antimicrobial agents
agains t fungi and bacteria caus e uterine contractions
and are s trongly antis eptic [6]. A bout four grams of
Syzygium aromaticum are boiled in three liters of water
until half the water has evaporated. This water, t a ken
in draughts , will s low down s evere s ymptoms of
cholera [1 8 ]. In general medicine, Syzygium aromaticum
Corresponding Author: Ghaly, M .F., Botany Department, Faculty of Science, Zagazig University, Egypt.
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J. App. Sci. Res., 5(10): 1298-1306, 2009
is us ed as an agent agains t flatulence, s tomach
dis tens ion and gas tro-intes tinal s pam[1 0 ]. Eugenol a main
cons tituent of the es s ential oil obtained from commonly
cons umed s pecies s uch as Pime n t a r a cemosa,
Cinnamomum verum and Syzygium aromaticum is us ed
as antis eptic, antibacterial, analges ic agent in traditional
me d ical practices [1 2 ]. Eugenol is a phenolic compound,
which it is the main component of clo v e plant, with
the antimicrob ia l d ata agains t is olated bacterial
organis ms . Caryophyllene has als o been s how n to
pos s es s antimicrobial properties , which obtained as
s econd cons tituents obtained from plant [3]. The aim of
th e pres ent s tudy to evaluate the antibiotics action
agains t b a c terial pathogens to UTI by us ing different
medicinal plant extract combined w it h antibiotics to
minimize the dos e and duration of antibiotics us ed.
Determination the protein ban d s of bacterial under
s tres s of antibiotic combined with the plant e xtract and
identified the mos t active s ubs tance purified from
active medicinal plant as eugenol.
MATERIALS AND METHODS
The pres ent s tudy is conduc t e d on 100 urine
s amples were taken from patie nts who were examinated
for UTIs in Zagazig Univers ity hos pitals (inpatients ) or
attending Zagazig Un ivers ity clinics (outpatients ),
Urology Department, thes e s amples were collected from
A pril 2007 to A u gus t 2007. A pos itive bacterial growth
on CLED , M acConkey's agar and nutrient agar, the
diagnos is of UTIs in urine s amples was bas ed on the
pres ence of $ 105 CFU o f microorganis ms per ml in
urine culture [7]. A ls o, pres ence of more than 5 pus cells
per high po w e r field in an uns pun urine in male, and
more than 10 p u s cells in a female, and is defined as
pyuria [2 6 ]. High number of p u s c e lls in urine, or pyuria,
us ually indicates infection.
Bacterial is olates were identified a c cording to the
ke y o f (Be rg e y 's M a n u a l) of Determin a t iv e
Bacterio logy [1 5 ]. A ll s trains of bacteria is olated were
tes ted for antimicrobial s ens itivity by s tandardized dis c
diffus ion technique, which was done as des crib e d by
Bauer et a l . [5]. Take one colony from each is olated
bacterial is olates by s terile loop th e n inoculated into 5
ml s terile nutrient broth and incubate for 24 h o urs at
37 o C, then make turbidity eq u a l 0.5 M c ferland
s tandard s alin (0.05 ml barium chloride + 9.95 ml
s ulfuric acid)[4]. Each broth inocula were applied b y
s terile s wabs on M uller-Hinton agar plates ; two pla t e s
were us ed fo r each is olates in which antibiotics dis cs
were applied to the s u rface of plates at cons tant
dis tances . The antibiotic s us ed in this experiment were
penicillin-G (P.G) (10µg), ampicillin (A M ) (10µg),
a zit h romycin (A ZM ) (15µg), norflo xa c in (N O R)
(10µg), ofloxacin (O F X ) (5µg), amikacin (A K) (30µg),
1299
chloramphenicol (C) (30µg), cefotaxime s odium (CT X)
(30µg). The p la t e s were incubated at 37 o C for 24h.
The inhibition zones were meas ured with a millimeter
ruler. The entire d ia meter of inhibition zone was
meas ured including the diameter of the dis c. The e nd
p o int of the reading was taken as complete inhibitio n
of the growth to the naked eye [2 9 ].
The antibiotics , ofloxacin, amikacin were s elected
according to s ens itivity of different bacterial is olates to
determine the M IC and M BC agains t tes ted bacterial
is o la t e s n a me ly E. coli, Klebseilla, P r o t e u s ,
Pseudom o nase and Staphylococcus, according to
Lowry,[2 2 ]. The medicinal plant extracts were prepared
according to Huang et al.,[1 6 ].
Bacterial protein
e xt raction was performed
according to Saxena et al.,[2 7 ], w h ere protein content
were extracted and d e t e rmined in treated bacteria
(combination between M IC of ofloxacin for each tes ted
o rg anis ms and extract of Syzygium aromaticum) and
control (non-treated) bacteria.
Spectros copic analys is of the purified antimicrobial
s ubs tances obtained from Syzygium a r omaticum by
TLC method was performed by M icroanalytical Center
of Cairo Univers ity, Egypt. Th e infra-red (IR) s pectrum
of the active fraction wa s d e termined in potas s ium
bromide (KBr)[2 1 ]. Infra-red s pectrophotometer is model
PYE Unicum SP1100 USA . A ls o, the active frac t ion
was determined in Dimethyl s ulfoxide (DM S O ) us ing
n u c lear magnetic res onance (H-NM R)[2 8 ]. Nuclear
magnetic res onance is model Gemini-200 1 HNM R.
RES ULTS AND DIS CUS S ION
The urine s amples have been collected from
different ages o f males and females . The res ults in Fig.
(1) indicated that the number of contaminated urine
s amples collected from males an d females were 100
s amples , 40 contaminated with E. c o l i , 22 of Proteus
mirabilis, 15 o f Klebsiella pneomoniae, 13 of
Staphylococcus saprophyticus and 10 of Pseudomonase
aeru g inosa. So the highes t percentage of dis tribution
are found in E. coli (40.0%) follo w e d by Proteus
mirabilis (22.0%), Klebs i ella pneomoniae (15.0%),
S t a p h y l o c o c c u s s a p r o p h y t i c u s (13.0% ) a n d
Pseudom o n a s e aeruginos a (10.0%). Out of the (87.0%)
of all is olates that belong to Gram-n e g ative organis ms ,
Enterobacteriaceae cons titute the main grou p (77.0%),
while Pseudomonas aeruginosa w as res pons ible for
only (10.0%) of the infection. (13.0%) of the is olates
belong to the Gram-pos itive cocci; Staphylococcus
saprophyticus. M ady and Helmi,[2 3 ] found that 83.1% of
all is olates belong to Gram-negative organ is ms . Out of
thes e, Enterobacteriaceae c o ns titute the main group
(79.0% of all is olates ), while Pseudomonas a e r u ginosa
was res p ons ible for only (4.1%) of the infection.
J. App. Sci. Res., 5(10): 1298-1306, 2009
Fig. 1: The dis tribution number of pathogenic bacterial is olates from UTI patients .
(15.2%) of the is olates belong to the Gra m-pos itive
pathogens , and the mos t common was Entero coccus
s pp (9.0%). Candida spp. cons tituted only 1.7% of the
is olates ; thes e res ults were in agreement wit h our
pres ent res ults .
The s ens itivity tes t res ults agains t bacterial is olates
in Fig. (2) and photos No. (1) s hown t hat the antibiotic
ofloxacin is more effective agains t is olated pathogenic
bacterial organis ms , which the percentage of s ens itive
organis m reach to 58.0% follo wed by amikacin 54.0%,
c h lo ra mp h e n ic o l 52.0% , n o rflo xa c i n 5 1.0% ,
a zithromycin 48.0%, ampicillin 11.0%, cefot a xime
s odium 2.0% and penicillin-G 0.0%. The antibiot ic
a mikacin s how intermediate effect agains t is olated
pathogenic bacterial organis ms , which the percentage of
organis m re ach to 27.0% followed by azithromycin
13.0%, cefotaxime s odium 12.0%, chloramp h enicol
10.0%, ofloxacin 7.0%, norfloxacin 5.0%, ampicillin
1.0% and penicillin-G 0.0%. On the other hand, the
antibiotic penicillin-G has n't any effect again s t is olated
pathogenic bacterial organis ms , which the percentage of
res is tance organis m rea c h t o 100.0% followed by
a mp ic illin 88.0% , c e fo t a xime s odiu m 86.0% ,
n o rflo xa c i n 4 4 . 0 % , a z i t h r o m y c i n 3 9 . 0 % ,
chloramphenicol 38.0%, ofloxacin 35.0% and amikacin
19.0%. A deyemo and s ome colleagues reported that
while all urinary is olates were poorly s us ceptible to
t rimethoprim-s ulphamethoxazole and ampicillin, they
exhibited good s us c e p t ib ilit y t o nalidixic acid,
nitrofurnation and ofloxacin [2]. The pres ent s tudy
s howed that the s us ceptibility rat e of urinary is olates
was highes t for meronem (76.19%), followed by
amikacin (70.27%), nitrofurantion (66.60%), norfloxacin
(64.28%) and gentamicin (58.33%)[1]. Thes e res ults go
in line with our res ult s , where it des cribed that all
urinary is olates were poorly s us ceptible to ampicillin,
b ut exhibited good s us ceptibility to ofloxacin, amikacin
and norfloxacin.
Fifteen bacterial is olates and The mos t two
effective ant ib iotics were chos en to perform M IC and
M BC tes t, where the res u lt s in Table (1) indicated that
1300
the maximum M BC w e re obtained at ofloxacin
antibiotic which recorded 250 µg/ml agains t P.
aeruginosa number 96 & 42, K. pneumoniae number
93 and Proteus mirabilis number 43 & 55, and the
lowes t M BC were obtained 31.25 µg/ ml at E. coli
number 7 and Staphylococcus sapr ophyticus number
76. M BC equal to M IC which recorded 250 µg/ml &
125 µg/ml o f o floxacin agains t P. aeruginosa number
42 & E. coli number 65 res pect iv e ly. The res ults
indicated that t h e ma ximum M BC were obtained at
amikacin antibiotic which recorded 250 µg /ml agains t
E. c o l i n u mber 24, 65 & 72, P. aeruginosa number 42,
K. pneumo n i a e number 93 and Proteus mirabilis
number 47 & 55, and the lowes t M BC were obtained
62.5 µg / ml at E. coli number 7 and Staphylococcus
saprophyt i c u s number 9 & 76. M BC equal to M IC
which recorded 250 µg/ml of a mikacin agains t E. coli
n u mber 24, K. pneumoniae number 93 followed by P.
aeruginosa number 96, Pro t eus mirabilis number 43 &
45 and E. coli number 69 which re corded 125 µg/ml
followed by Staphylococcus saprophyticus number 9 &
76 which recorded 62.5 µg/ml.
A n attempt was made to tes t the antagonis tic effect
of different medicin a l plant extracts (ros emary, orange
peel, ga rlic, lemon gras s , peppermint, s pearmint,
h ib is c u s , ma rjoram, thyme, tilia, clove, fennel,
cinnamon, cas tor plant, ginger a n d chamomile), as cold
water extract, boiled water extract and a lc o h olic extract
agains t fifteen c lin ical bacterial is olates . The res ults in
Fig. (3) and photos No. (2) illus trated that ros emary,
le mon gras s , peppermint, hibis cus , clove, marjoram,
thyme, cinnamon, cas tor plant and chamomile were
mos t effective plants extracts agains t s elected pathogens
(Escherchia coli, Klebsi e l la pneumoniae, Proteus
m i r a b i l is, Pseu d o mo n a se a eru g in o s a a n d
Staphyloccocus saprophyticus). However in three cas es
(cold water, boiled w a ter and alcohol extract), ginger
was the lowes t effective plant extracts . Orange peel and
fe n nel were mos t effective only in cas e of alco h o l
extract, low effective in cas e of boiled w a t e r extract,
a n d n o t effective in cas e of cold water extract. M ore
over
J. App. Sci. Res., 5(10): 1298-1306, 2009
Fig. 2: The s ens itivity tes t of pathogenic bacterial is olates agains t different antibiotics drugs .
Photos No. 1:
A ntibiotic s us ceptibility of bacterial is olates by dis c diffus ion method.
A K = amikacin; PG = penicillin -G; CTX = cefotaxime s odium; OFX = ofloxacin; NOR =
norfloxacin; A M = ampicillin; A ZE = azithromycin & C = chloramphenicol.
M i n i mum inhibitory concentrations (MICs) (µg/ml) and minimum bactericidal concentrations (MBCs) of ofloxacin and ami k aci n
antibiotics.
Parameters
Ofloxacin
Amikacin
--------------------------------------------------------------------------------------Bacterial isolates No.
(MIC) (µg/ml)
(MBC) (µg/ml)
(MIC) (µg/ml)
(MBC) (µg/ml)
E. coli 7
7.82
31.25
31.25
62.5
S. saprophyticus 9
7.82
62.5
62.5
62.5
E. coli 24
62.5
125
250
250
P. aeruginosa 42
250
250
125
250
P. mirabilis 43
125
250
125
125
P. mirabilis 45
62.5
125
125
125
P. mirabilis 47
62.5
125
125
250
P. mirabilis 55
125
250
125
250
E. coli 65
125
125
125
250
E. coli 69
62.5
125
125
125
E. coli 72
62.5
125
125
250
S. saprophyticus 76
7.82
31.25
62.5
62.5
P. mirabilis 85
15.63
62.5
62.5
125
K. pneomoniae 93
125
250
250
250
P. aeruginosa 96
125
250
125
125
Table 1:
garlic was the mo s t effective in cas e of cold
water and alcohol extract, a n d n ot effective in cas e
of boiled water extract. Spearmint and tilia haven't
any effect a g a in s t bacteria. However in three
cas es (cold water, boiled water and alco h o l e xtract).
The pres ent s tudy illus trates that thyme, ro s e mary,
1301
caraway
and
dianthus are mos t effectiv e plant
extracts agains t s elected pat h ogens (E. coli, K.
aerogense, P. aeruginosa). In two c a s e s , water
and a lc o hol extract. Clove extract had potent
a n t imicrobial
activity
agains t the entire Gramnegative
J. App. Sci. Res., 5(10): 1298-1306, 2009
Fig. 3: Sens itivity of bacterial is olates agains t cold water, boiled water and alcoholic plant extracts .
Photos No. 2: Sens itivity of bacterial is olates agains t cold water, boiled water and alcoholic plant extracts .
1- Ros emary, 2- Chamomile, 3- Thyme, 4- Cinnamon, 5- Ginger, 6- Orange pe e l, 7- Peppermint,
8- M a rjo ra m, 9- Fennel, 10- Lemon gras s , 11- Cas tor plant, 12- Hibis cus , 13- Tilia, 14- Spearmint,
15- Garlic, 16- Clove.
1302
J. App. Sci. Res., 5(10): 1298-1306, 2009
and Gra m-pos itive organis ms tes ted (S. aureus,
Enterococcus cloacae, Salmonella paratyphi, Klebsiella
pneumoniae, E. coli, C i t robacter spp, and Candida
albicans [3].
The res ults in Tables (2 and 3) and photos No. (3,
4) illus trated that there is s ynergis m between the
combination of clove extract and antibiotics (ofloxacin
and amikacin), but there is n't s ynergis m between the
combination of garlic extract and antibiotics (ofloxacin
and amikacin). This s tudy with agree with our s tudy
where it illus trated that clove extract s how s ynergis m
with ofloxacin but no s yn e rg is m between garlic extract
and ofloxacin [1 7 ].
The clinical bact e ria l is olates P. aeruginosa 42, P.
mirabilis 55, K. pneumoniae 93 we re g row on nutrient
a g a r as control and on nutrient agar with antibio t ic
ofloxacin with concentrations (125, 62.5 & 62.5) µg/ml
res pectively combined with clove extract (boiled water
extract) to determine protein band at control and treated
bacteria, the res ults in photo (5) indicated that there are
many protein bands induced a n d other dis appeared if
compared with control.
The res ults obtained from s pectrum analys is
1
HNM R of compound s howed s ignals at 3.8(d, 2H,
CH2 ), 5(t, 2H, =CH2 ), 6(m, 1H, -CH=), 6.5-6.8(m, 3H,
A r H's ) and at 8.6(S, 1H, OH); as illus trated in Fig.
(4). IR of compound s howed broa d band at 3500 cm-1
corres ponding to –OH gro u p and band at 1518 cm-1
corres ponding to C=C, in addition to O-C band
a b s o rp tion at 1099 cm-1 ; as illus trated in Fig. (5).
Thes e data corres ponding to eugenol s tructure.
Table 2: Combination effect between different medicinal plant extracts and MICs of ofloxacin against bacterial isolates
Bacterial isolates No.
Ofloxacin MICs
Diameter of inhibition zones (mm) of medicinal plant extracts
----------------------------------------------------------------------------------------------------------------------------------------Rosemary
Garlic
Clove
Castor plant
Peppermint
Hibiscus
µg/ml
IZ
IZ
IZ
IZ
IZ
IZ
IZ
E. coli 7
7.8125
12
14
12
13
12
12
12
S. saprophyticus 9
7.8125
12
10
11
15
ND
ND
15
E. coli 24
62.5
12
15
ND
18
13
14
15
P. aeruginosa 42
250
15
17
15
18
11
15
17
P. mirabilis 43
125
15
15
12
23
ND
25
20
P. mirabilis 45
62.5
12
22
15
20
ND
17
20
P. mirabilis 47
62.5
12
ND
12
18
12
13
16
P. mirabilis 55
125
15
15
12
20
11
20
22
E. coli 65
125
14
17
12
15
14
13
18
E. coli 69
62.5
11
15
ND
19
ND
15
14
E. coli 72
62.5
15
ND
16
17
11
10
12
S. saprophyticus 76
7.8125
15
15
18
18
17
11
23
P. mirabilis 85
15.625
15
10
12
20
ND
10
20
K. pneomoniae 93
125
13
23
10
20
23
21
20
P. aeruginosa 96
62.5
12
14
12
18
10
15
14
Table 3: Combination effect between different medicinal plant extracts and MICs of amikacin against bacterial isolates.
Bacterial isolates No.
Amikacin MICs
Diameter of inhibition zones (mm) of medicinal plant extracts
------------------------------------------------------------------------------------------------------------------------------------------Rosemary
Garlic
Clove
Castor plant
Peppermint
Hibiscus
µg/ml
IZ
IZ
IZ
IZ
IZ
IZ
IZ
E. coli 7
31.25
12
12
16
15
12
12
14
S. saprophyticus 9
62.5
12
15
10
16
12
14
13
E. coli 24
250
12
11
11
20
13
14
13
P. aeruginosa 42
125
10
14
12
15
11
14
18
P. mirabilis 43
125
12
15
12
19
19
14
13
P. mirabilis 45
125
10
12
14
15
13
12
15
P. mirabilis 47
125
11
12
11
18
13
14
22
P. mirabilis 55
125
13
13
10
18
22
15
12
E. coli 65
125
13
16
ND
15
17
15
15
E. coli 69
125
10
13
ND
17
10
12
20
E. coli 72
125
12
15
ND
15
ND
10
14
S. saprophyticus 76
62.5
13
22
26
25
15
20
27
P. mirabilis 85
62.5
10
14
10
18
ND
12
17
K. pneomoniae 93
250
13
15
ND
16
15
12
20
P. aeruginosa 96
125
15
18
12
19
13
12
21
T he pres ent s tudy illus trates that the compound
res pons ible for the clove aroma is eugenol. It is the
main component in the es s ential oil extracted from
1303
cloves , compris ing 72-90%. Eugenol h a s pronounced
antis eptic, antimicrobial and anaes thetic properties [9].
J. App. Sci. Res., 5(10): 1298-1306, 2009
Photos No. 3: Combina t io n b e t w e en medicinal plants extracts and ofloxacin antibiotic s us ceptibility of bacterial
is olated by dis c diffus ion method.
Photos No. 4: Combination between medicin a l p la n t s e xtracts and amikacin antibiotic s us ceptibility of bacterial
is olated by dis c diffus ion method.
1- Garlic, 2- Clove, 3- Ros emary, 4- Peppermint, 5- H ib is cus , 6- Cas tor plant, 7- M IC of ofloxacin
or amikacin.
Photos No. 5: Protein analys is for clinical bacterial is o la t e s b e fo re a n d after s tres s by comination between
ofloxacin and clove extract
1- M arker. (26.000, 45.000, 66.000, 85.000, 96.000, 105.000) KD. 2- Pseudomonas aeruginosa
number 42 before s tres s . 3- Pseudomonas aeruginosa number 42 after s t re s s . 4- Proteus mirabilis
n umber 55 before s tres s . 5- Proteus mirabilis number 55 after s tres s . 6- Klebs iella pneumonia e
number 93 before s tres s . 7- Klebs iella pneumoniae number 93 after s tres s .
1304
J. App. Sci. Res., 5(10): 1298-1306, 2009
Fig. 4: 1 HNM R s pectrum of the antimicrobial s ubs tances .
Fig. 5: IR s pectrum of the antimicrobial s ubs tances .
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