INTRODUCTION:
Rhizosphere microbiology has determined
significant consideration
through the last century
because of the role played in plant growing and health. P.aeruginosa are
ubiquitous bacteria that are ordinary resident of the plant
rhizosphere and are the mainly studied group with in the genus Pseudomonas (1).
the biocontrol ability of these strains is directly associated with the
production of antibiotics such as 2-4-diacethyl-phloroglucinol (DAPG),
Pyrrolnitrin (PRN), Phenazines (Phz), hydrogen cyanide (HCN) and Pyoluteorin
(PLT) (2).
These antibiotics have been described to exhibition antibacterial, antifungal, antihelmenthic and phytotoxic
activity(3). Most of these antibiotics were secondary metabolite manufactured
during the log phase when bacterial growing is limited by the exhaustion of any
one of the significant nutrient source. These metabolites are chemically and
functionally various with notable antimicrobial, plant growth regulatory and
plant enzyme inhibitory (4). One of these compound pyoluteorin is aromatic
phenolic polyketide antibiotic that was first separated from P.
aeruginosa and later from P. fluorescens. These compounds have
been examined intensively because their broad spectrum antibiotic properties
and roles in virulence, against various bacterial and eukaryotic species (5). The aim of this study was isolated P. aeruginosa
from the rhizospher of wheat plant and studied the production of pyoluteorin
antibiotic. Extracted and Purification and characterization by using (TLC), (HPLC),
( FTIR),and Studying the antimicrobial activity of crude and purified
pyoluteorin against UTI isolatesbacteria and candida .as well as prevention formation
of biofilm of some MDR UTI isolates.
MATERIAL AND METHODS:
Isolation and Identification of P.aeruginosa
isolates:
The Soil samples were taken from rhizosphere areas
from wheat plants as mentioned in (6) Bacterial isolates were subjected to number of cultural and
biochemical tests for identification proposed by (7).
Isolation andIdentification of UTI bacteria and fungi:
Bacteria and fungi were isolated from Urine specimen
collected from local hospitals in Baghdad city, Identification of isolated were doneaccording to the
morphological and biochemical test (8). VITEK 2 system were used to confirmed
the identification of all isolates.
Extraction of pyoluteorin:
The pyoluteorin production by isolate of P.
aeruginosa PA3 was obtained as described by (9). The isolate PA3 was pre
cultured in King’s B broth at 28 ºC for 12 h then transferred to pigment
production medium ( PPM) .The cultures were incubated in a rotary shaker for 4
days at 110 rpm. For extraction centrifuged at 12000 rpm for 10 minutes
acidified to pH 2 with 1N HCl and mixed with the same volume of ethyl acetate.
The crude PLT was obtained riedina desiccated vacuum at 40°C and then Dissolved
in 1ml methanol.
Purification of pyoluteorin:
Column Chromatography:
The first step in purification of extracted PLT was
carried out using gel filtrerationchromatography ,1ml of extracted PLT was
placed on silica gel column (1.5*25 cm) the column was eluted using solvent
Chloroform and acetone from 9:1 (v/v) elution flow rate was 1ml/min (10). The crude separate to be fractionated was added on the silica gel
column surface and the extract was adsorbed on top of silica gel.
Thin layer chromatographic method (TLC):
The second step in purificationpyoluteorinis TLC was used as described by.(11) with some modifications
as follows: - The sheet (silica gel 60f-254, 0.2 mm, layer thickness and aluminum
support, size 20×20 cm, Spain) was used for analyzing samples. Positioning line
was marked 1 cm from bottom edge of the plate. Twenty microliter from the
fraction of gel filteration of silica gel column samples was applied to
thin-layer chromatography plates coated with a 250 ml layer of silica gel and
developed in Chloroform and aceton (9 :1 v/v) as solvent system. The spots were
visualized by spraying with diazotized sulphanilic acid or under UV at 254 nm
in an UV illuminator. Then the spotScrubbed by the spatula and dissolved in ethyl acetate solvent finally dry in a desiccated vacuum at 40°C
and then dissolved in methanol. Rf values of the spots were compared with
synthetic antibiotics Rf=0.5.
High performance liquid chromatography (HPLC):
Partial pure of pyoluteorin were investigated on
preparative HPLC column and the chromatogram. 70% methanol was used as the
mobile phase, the sample was loaded on a HPLC (Shimadzu LC-8A, Kyoto, Japan)
C18 reversed-phase column (Zorbax SB-C18, 5.0µm, 4,6 mm*250 mm, Rockland
Technologies Ind., Newport, DE, U.S.A.), and the column was eluted with
methanol-water [70:30, v/v] at a flow rate of 2.0 ml min-1werepyoluteorin
detected by UV at 308 nm and determined the retention times of compound which
is specific value for each compound. The bioactive pyoluteorin purification was
pooled and its HPLC profile was compared with peak of partial purified HPLC
profile. (12).
Antibacterial activity of crude and purified
Pyoluteorin on uropathogenic bacteria:
A-Effect of crude pyoluteorin (PLT) on uropathogenic
bacteria: The agar well diffusion method was used to distinguish the action of
crude PLT produced from p.aeruginosa PA3 against uropathogenic bacteria
and Candida albicans.
B-Effect of purified PLT on uropathgenic isolates:
It was made as described previously in A but instead
of crude PLT, thewells filled with 100µl of purified PLT in different
concentration (50,100,200), µg/ml.
Biofilm formation by tissue culture plate method:
A-Biofilm
production of the some isolates of uropathogenic bacteria (12 isolates of K.
pneumoniae and 10 isolates of S. aureus) was determined by a
modification of the method tissue culture plate described by (13) and (14). B-
Inhibition of biofilm formation by pyoluteorin.: It was done asmentioned previously
in A- but Aliquots of 100 µL of the culture and 50 µL ofPLTwere loaded to
microplates and incubated for 24 h/37◦C.
RESULTS AND DISCUSSION:
Extraction of crude PLT with ethyl acetate from the Isolate
P.aeruginosa PA3:
In this experiment extracted pyoluteorin with ethyl
acetate as organic solvent, produced from P.aeruginosa PA3 which
appeared highly activityon filtrate against uropathogenic isolates. The organic
phase was separated dried then resuspended with methanol the compound obtained
was crude pyoluteorin.
Purification of
pyoluteorin from P. aeruginosa PA3:
1- Column
chromatography:
Crude extract was subjected to silica gel column (1.5 X 25) cm and 60- 120 mesh size) 1ml/min flow rate. The compound was eluted using solvent ratios from 9:1
of chloroform and aceton, (10). Crude extract to be
fractionated on the silica gel column surface and the extract was adsorbed on
top of silica gel. The
fractions extracts were collected by class tube and the Partial purification of
antibiotic compound were eluted on HPLC.
High Performance Liquid Chromatography:
Partially purified compound PLTwereinvestigated on
preparative HPLCcolumn and the chromatogram were shown in Fig.(1) When
methanol-water(70:30, v/v) was used as the mobile phase, the results obtained showed
there was two peaks appeared on fig (1) first one belong to the solvent on
retention time 3.85. While the second in Rt= 7.04 belong to PLT with low
concentration.
Figure 1: HPLC for separation pyoluteorin onConditions:
C-18 column (250 mm×4.6 mm, 5µm); flow rate: 2.0ml/min; Detection wavelength:
308nm; 70% methanol
2-Purification of Pyoluteorin through TLC:
The active fraction obtained from column silica gel
exposure to TLC and there results in Figure (2) showed that the pyoluteorin was
produced by isolate PA3 by giving a band with RF 0.50.Pyoluteorin antibiotic
detection was prepared through TLC based on mobility of the compound on the
silica plate that is measured in terms of retardation factor. The Rf value of
0.50 corresponded to pyoluteorin antibiotic with Chloroform : Acetone solvent
system and confirmed the expression of PLT genes. The pyoluteorin
antibiotic detected on the based at RF value (0.50) by (15), (16) and (17). The
obtained results were found similar to those detected by (18) who use TLC
assessment to purify pyoluteorin with RF 0.50. (19) used TLC technique for
identification of pyoluteorin produced by P. aeruginosa as antifungal
compounds, and (18) used TLC as initial step for isolation and analysis of
antibacterial substance produced by P. aeruginosa.
Figure 2: Detection of pyoluteorin produce by
P.aeruginosa under A-Visible light B-Under UV light
3-Identifiction of pyoluteorin by HPLC:
The spots found on TLC plate at RF=0.5 were scarped as
mentioned previously and loaded on a HPLCcolumn. The obtained highest active
fraction was analyzed by HPLC. sample was loaded on a HPLC (Shimadzu LC-8A,
Kyoto, Japan) Elution conditions of purification were investigated on HPLC
columnthe chromatogram were shown in Fig. (3), the peaks of purified PLT shown
the retention time were 7.09 min. The results showed there was single peak
belong to PLTRt=7.09 with high concentration.
Figure 3: HPLC Chromatogram for Plt purification.
Conditions: C-18 column (250 mm×4.6 mm, 5 µm); flow rate: 2.0 mL/min; detection
wavelength: 308 nm; 70% methanol.
The Present study showed that purification steps used
in these experiment with extracted crude pyoluteorin very effect comparing with
the results of others researchers using the same condition in purification by
silica gel column the peaks obtained in HPLC (with certain condition)in
retention time 7.04 (and these is the retention time specific for pyoluteorin
in same condition). And in the follower steps by TLC and the product found in
RF=0.5 which is specific for pyoluteorin indicated by many researchers by using
the same experimental condition and solvent when using HPLC at the last steps
of purification the retention time of the peaks obtained was 7.09 with highly
concentration as it appeared in figure(3). Characterization of pyoluteorin
showed the molecule was yellow aromatic polyketide antibiotic can soluble in
chloroform, dichloromethane, water, and can increase solubility when increasing
temperatureand that is agreementwith the characterpointed by (20). (21).
Antimicrobial activity of pyoluteorin:
Antimicrobial activity of crude and purified
pyoluteorin on some UTI bacteria using agar diffusion method against one
selected isolates of each uropathgenic isolatesthat showed higher resistance to
antibiotic the result summarized on table (1). Results showed that highest
activity of crude pyoluteorin appeared on Streptococcus agalactiae bacteria
34mm and the lowest activity was recorded on Enterobacter cloacae (8mm)
compared with purified pyoluteorin. Different concentration of purified
pyoluteorin, produced by isolate PA3, included (50, 100 and 200µg/ml) were
prepared to the activity determine of pyoluteorin against different
microorganisms included: Gram-positive such as Streptococcus agalactiae,
Staphylococcus aureus. Gram-negative such as Escherichia coli, Proteus
spp, Acinetobacterspp and Enterobacterspp and yeast as Candida albicans Results
indicated that pyoluteorin antibiotic shows high antimicrobial activity against
all microorganisms. The concentration pyoluteorin 200 μg/ml of purified
pyoluteorin was found to have higher activity for isolate PA33.The inhibition
zone of PLT on Staph.aureus34, 32, 30, mm in concentration 200, 100, 50, µg/ml
respectively and for E.coli the inhibition zone was 20, 10, 10, respectively.
While in candida the I.Z were 25, 15, 10 respectively. Many studies reported
that P.aeruginosa could produce various secondary metabolic which could
playan important role in controlling pathogens and could produce abroad
spectrum bacterial and fungicidal compound and pyoluteorin was one of these
compound (22)
Biofilm production:
Bacterial biofilms play an essential role in UTI
infection as they responsible for persistent of infections and important to
recurrences and relapses (23). Biofilms production was detected in 22
Uropathogenic organisms by Tissue Culture Plate (TCP) method of two species 10
strain of Staphylococcus aureus and 12 isolate of Klebsiella pneumoniae.
The results of biofilm production of staphylococci by TCP method appeared
that biofilm production was detected in 8 (80%) of the 10 staphylococcal
isolates with different intensities; 4 (40%) isolates were strong producers,
3(30%) isolates were moderate and 1 (10%) isolates were weak biofilm producers,
whereas 2 (20%) were non biofilm producersand biofilm production of Klebsiella
by TCP method was detected in 10(83%) of the 12 Klebsiella isolates with
different intensities 4(33.33%) isolates were strong producers, 3(25%) isolates
were moderate and 3 (25%) isolates were weak biofilm producers, whereas 2
(16.66%) were non biofilm producers (Table 2) (Figure4) .
Tabel 1: Inhibition Zone
of crude and purified pyoluteorin extracted from P.aeruginosa PA33 agains UTI isolates
|
Zone
of inhibition of purified pyoluteorin (µg/ml)
|
Zone
of inhibition (mm)
Crude
pyoluteorin
|
Bacterial
isolates
|
|
200
|
100
|
50
|
|
12
|
0
|
0
|
16
|
Klebsiella
pneumonia
|
|
20
|
10
|
10
|
10
|
Escherichia
coli
|
|
12
|
10
|
10
|
15
|
Pseudomonas
aeruginosa
|
|
20
|
17
|
12
|
12
|
Serratiamarcescens
|
|
14
|
12
|
0
|
8
|
Enterobacter
cloacae
|
|
20
|
12
|
0
|
12
|
Acinetobacterspp
|
|
17
|
15
|
12
|
15
|
Proteus
mirabilis
|
|
14
|
12
|
10
|
12
|
Morganellamorganii
|
|
34
|
32
|
30
|
30
|
Staphylococcus
aureus
|
|
24
|
20
|
15
|
34
|
Streptococcus
agalactiae
|
|
25
|
13
|
10
|
24
|
Candida
albicans
|
Figuer (4) K.pneumoniae biofilm formation by
tissue Culture
Plate Method and inhibition biofilm by pyoluteorin
antibiotic Inhibition
of biofilm formation by pyoluteorin:
The anti-biofilm activity of pyoluteorin antibiotic
was also investigated in the prevention of biofilm formation by analyzing the
biofilm cells viability and by quantifying biofilm biomass the best outcomes
achieved most of the PLT produced synergic outcomes with high reductions often
with almost or total absence of biofilm-cell viability. The biofilm was
obtained and its mass quantified using the crystal violatetechnique (24).
The result of anti-biofilm production from K.pneumoniae and S.aureus
strains as showed in figure (4) and table(3) 6 isolates of S.aureus show
none biofilm production, 3 strain show weak biofilm produced and 1 strain shows
moderate biofilm product and 5 strains of K.pneumoniae showed none
biofilm product and 7 strains weak biofilm product.
Table 3:inhibition biofilm formation by pyoluteorin antibiotic
|
Antibiofilm
|
S. aureus
No.=10
|
%
|
K.pneumoniae No.=12
|
%
|
Totale
No.=22
|
%
|
|
Strong
|
0
|
0
|
0
|
0
|
0
|
0
|
|
Moderate
|
1
|
10
|
0
|
0
|
1
|
5
|
|
Weak
|
3
|
30
|
7
|
58
|
10
|
45
|
|
None
|
6
|
60
|
5
|
42
|
11
|
50
|
|
Total
|
10
|
100
|
12
|
100
|
22
|
100
|
Numerous study focusing on
the mode of action by which antimicrobial compound could controlling pathogenic
agents. (22).(25.), others studies pointed the important of
biofilms as virulence factors ,so by prevention the biofilms
formation,pyoluteorin could be consider one of novel antibiotic in treatment
various bacterial infections.
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