Iranian Journal of Fisheries Sciences
17(1) 162-178
2018
DOI:10.22092/ijfs.2018.115592
Comparison of antibiotics and bacteriocins antibacterial
activity on Xanthomonas citri subsp.citri
Gholami D.1, 2; Aminzadeh S.1*; Alavi S. M.1; Kazemipour N.3;
Ghoroghi A.4; Emruzi Z.1.
Received: April 2016
Accepted: July 2016
Abstract
Citrus canker is a citrus disease species created by the bacterium Xanthomonas citri
subsp.citri. Many citrus, such as oranges, lime, and grapefruit are affected by the
infectious bacteria and stems, leaves and fruits are experiencing loss. In this experiment
antibacterial effects of five kinds of antibiotics including ampicillin, kanamycin,
chloramphenicol,
penicillin,
streptomycin,
Cronobacteriocin
DGH2
and
Enterobacteriocin DGH4 were evaluated on 107 strains of Xanthomonascitri
subsp.citri. MIC and MBC data for antibiotics and bacteriocins against Xanthomonas
strains were performed. According to this project, Xanthomonas strains were
comparatively susceptible and resistant to Cronobacteriocin DGH2, Enterobacteriocin
DGH4, ampicillin, kanamycin, chloramphenicol, penicillin and streptomycin. NIGEB183 strain is the most sensitive to these antibiotics and bacteriocins. However, only the
NIGEB-242R1 strain is resistant to chloramphenicol. Penicillin has minimum
inhibitory effects on Xanthomonas strains. Based on this case study, chloramphenicol is
the most antibacterial activity among antibacterial agents and this compound is a good
candidate for inhibitory activity. Cronobacteriocin DGH2 has a moderate antibacterial
activity against Xanthomonas strains.
Keywords: Antibiotics, Bacteriocins, Citrus canker, MIC and MBC data, Xanthomonas
citri subsp.citri
1-Department of Bioprocess Engineering, National Institute for Genetic Engineering and
Biotechnology, Tehran, Iran
2-Institute of Biochemistry and Biophysics (IBB), University of Tehran, Iran
3-School of Veterinary Medicine, Shiraz University, Shiraz, Iran
4- Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research Education and
Extension Organization (AREEO), P.O. Box: 14965/149. Tehran, Iran.
*Corresponding author's Email: aminzade@nigeb.ac.ir
163 Gholami et al., Comparison of antibiotics and bacteriocins antibacterial activity on …
Introduction
Citrus canker is a pandemic disease that
led to a severe economic downturn in
the citrus industry (Gottwald et al.,
2009; Behlau et al., 2010; Chen et al.,
2012). Disease symptoms consist of
water soggy ulcers that develop into
band scorch, then pimples and finally,
canker. Citrus canker can lead to
defoliation and immature fruit fall, in
severe cases (Brunings and Gabriel,
2003). The method of choice to control
the disease is sup plantation of putrefied
plants where it is not yet endemic. In
cases where the disease is endemic,
control is attempted by seeding trees
that are not patient, limiting the
extension between orchards, and using
preventive copper sprays (Graham et
al., 2004). However, none of these
actions control citrus canker efficiently
(Leandro et al., 2010). Antibiotics are
the most successful family of medicines
to protect human health and plants
(Stepanauskas, 2006). Because of the
emergence and dissemination of
antibiotic
resistance
and
their
association with continuous use of
antibiotics,
therapies
based
on
antimicrobial peptides are attractive
candidates as valid alternatives to
antibiotic treatments (Chen et al.,
2012). Bacteriocins are bacterial
ribosomally
synthesized
and
extracellular peptides or proteins with
an antibacterial activity usually against
bacteria intimately related to the
producer (Morisset and Frère, 2002;
Opsata et al., 2010; Castro et al., 2011;
Tashakor et al., 2017). These
antimicrobial therapies offer additional
advantages over drug therapies
currently used, because bacteriocins are
considered
as
natural
bioactive
compounds. Multiple aspects discern
bacteriocins from antibiotics; (i)
Bacteriocins are produced on the
surface of ribosomes in microbial cells,
while
antibiotics
are
primarily
secondary metabolites of the cell; (ii)
Bacteriocin producers are insusceptible
to the bactericidal agents, unlike
producers
of
antibiotics;
(iii)
Bacteriocins can attach to the target cell
wall anyplace on the surface, as no
specific receptors on the target cell wall
apparently exist; (iv) The mechanism of
bacteriocin on target cells is diverse and
is associated with the method of pore
formation in the outer cell membrane.
Bacteriocins bind to cell walls of
sensitive microbes, motive ionic
imbalances,
and
produce
pores
(Morisset and Frère, 2002). Inorganic
ions leak the target cells through the
created pores and thereby killing them.
Antibiotics, on the other hand, can
inhibit synthesis of the subcellular
processes
(cell
wall
synthesis,
intracellular protein production, and
DNA and RNA replication) (Svetoch et
al., 2011). Their bactericidal and
bacteriostatic mechanisms are diverse
and may comprise pore formation,
degradation of cellular DNA, disruption
via specific cleavage of 16S rRNA, and
blockage of peptidoglycan synthesis
(Todorov et al., 2011a). In this study,
the
inhibitory effect
of
both
Cronobacteriocin
DGH2
and
Iranian Journal of Fisheries Sciences 17(1) 2018
Enterobacteriocin
DGH4
also
antibiotics
including
ampicillin,
kanamycin,
chloramphenicol,
penicillin, and streptomycin over 107
strains of Xanthomonas were examined
and
the
inhibitory
power
of
antibacterial agents were compared
with each other on Xanthomonas.
Diffusion assays used to determine the
sensitivity of organisms isolated from
clinical samples have their limitations;
when two-sided outcomes are obtained,
the quantitation of antibiotic activity
needs to be more precise versus the
pathogen. As well as the designation
‘Resistant’ and ‘Susceptible’ may have
a realist explanation. So until in doubt,
detailed assessment method is to
determine the MIC of the antibiotic to
the organisms. In addition, the identical
tubes which can be used to test the
MBC as well have the added value
benefit (Adel et al., 2015).
Materials and methods
Bacterial strains and media
Two strains of Cronobacter sp. DGH1
and Enterobacter sp. DGH3 bacterium
were isolated from citrus orchards in
Sistan and Baluchestan region and was
screened for bacteriocin production
against indicator strains Xanthomonas
citri subsp.citri following previously
described method (Gholami et al.,
2014; Gholami et al., 2014b; Gholami
et al., 2015). All the producer and
indicator strains were cultured in
Nutrient Broth (Maryam et al., 2011;
Pragalaki et al., 2013) and Nutrient
Agar (Foltz and Martin, 1938) medium.
164
The bacteria against which the
antimicrobial
spectrum
of
Cronobacteriocin
DGH2
and
Enterobacteriocin DGH4 was studied
are enlisted in Table 1. All the bacteria
were procured from bacterial collection
of NIGEB, Iran.
Bacteriocin production
Nutrient broth was inoculated with an
18h culture (2%, v/v) of bacterial
strains and moved to shaker incubator
with 180 rpm at 37ºC (Svetoslav et al.,
2010). After cultivation of bacteria,
cells were removed by centrifugation
(10,000×g for 30min). The supernatant
was filtered through a center glass filter
with 0.2µm, pore size (Minisart, 17597,
Sartorius, Germany). The bacteriocin
present in the cell free supernatant
fraction by Amicon® ultra-15 PL-5 was
concentrated.
Bacteriocin enzymatic digestion test
Culture supernatant was incubated at
37ºC for 1h in the presence of various
enzymes (proteinase K, trypsin, papain,
pepsin) at a final concentration of 1 mg
mL-1 and 10 mL of each supernatant
was spotted onto soft agar inoculated
with 250 µL of 0.5 Mac Farland precultivated target strains. The plates
transferred at 32ºC and inhibition zones
were recorded.
165 Gholami et al., Comparison of antibiotics and bacteriocins antibacterial activity on …
Table 1: Antimicrobial1 agents effect on Xanthomonas citri subsp.citri strains.
Sample
AMP KAN
CHL
PEN
STR
CRO
ENT
112
12
15
8
10
5
7
NIGEB-26
9
12
18
9
10
5
3
NIGEB-88
8
10
13
8
7
2
2
NIGEB-201
6
10
12
10
6
5
1
NIGEB-88R
4
10
8
6
10
4
3
NIGEB-197
2
11
13
7
4
2
2
NIGEB-112
4
11
10
6
10
7
8
NIGEB-3369
9
10
13
8
9
2
2
NIGEB-204
4
10
6
6.5
8
11
7
NIGEB-K32
2
4
6
3.5
3
NIGEB-196
5
7
9
5
8
3
1
NIGEB-126
6
5
7
5
NIGEB-170
5
2.5
1
NIGEB-142
2.5
5
2.5
3.5
2
NIGEB-242R1
5
5.5
8
7
6
4
1
NIGEB-87
5
6
7
5
7
NIGEB-224
6
5
8
7
6
2
NIGEB-52
4
3.5
6
4
5
NIGEB-162
5
3.5
7
4.5
5
1
4
NIGEB-262sc1
5
5
7
5
4
4
2
NIGEB-260R2
4
5
8
7
5
NIGEB-157
4
2.5
6
5
4
NIGEB-286R1-2
4
5
8
5
5.5
1
NIGEB-243R1
5
3
7
3.5
3
NIGEB-244R1
5
4
6
3
3.5
2
NIGEB-147-1
4
5
9
6
5
3
NIGEB-153
3
4
7
6
7
4
2
NIGEB-249R2
2
2.5
5
3
NIGEB-136
6
5
8
8
6
3
1
NIGEB-213R2
6
5
11
10
8
NIGEB-54
5
6
10
8
7
2
NIGEB-7
5
6
9
5
4
NIGEB-211R1-1
5
6
6
6
4
2
NIGEB-247R1
6
6
9
8
7
NIGEB-10
6
8
9
8
9
2
2
NIGEB-53
7
6
10
8
8
NIGEB-313R1
7
8
10
8
9
NIGEB-232
2.5
3.5
7
4
3
2
NIGEB-96
8
7
9
8
10
2
1
NIGEB-246
5
12
18
9
12
1
NIGEB-13
7
5
16
10
2
3
NIGEB-180
9
6
17
7
6
2
3
NIGEB-186
9
11
15
9
8
2
NIGEB-259R1
8
6
14
10
8
3
NIGEB-75
8
16
15
5
11
NIGEB-79
12
8
18
10
8
4
NIGEB-22
6
7
16
3
8
3
3
NIGEB-264R1
11
10
20
10
4
2
1
NIGEB-94
4
4
6
4
NIGEB-112
5
7
11
4
3
NIGEB-81
8
5
10
11
5
6
5
NIGEB-287R2
3
4
11
6
6
3
3
NIGEB-138
9
5
12
11
5
1
NIGEB-227
6
7
11
5
6
2
3
NIGEB-36
10
11
17
8
13
5
3
NIGEB-16
10
15
20
14
14
4
NIGEB-168
Iranian Journal of Fisheries Sciences 17(1) 2018
Sample
AMP KAN
CHL
PEN
STR
CRO
ENT
6
12
18
12
15
2
2
NIGEB-169
8
11
16
10
4
1
NIGEB-65
17
7
13
20
4
NIGEB-173
2
4
13
3
11
4
5
NIGEB-98
11
10
18
10
2
2
1
NIGEB-127
15
7
10
20
10
2
3
NIGEB-184-1
6
7
NIGEB-184-2
7
2
13
4
10
NIGEB-122
5
9
12
5
10
1
2
NIGEB-134
7
2
12
8
13
7
4
NIGEB-125
10
9
15
13
8
NIGEB-71
2
6
NIGEB-116
5
8
11
9
NIGEB-263R1
3
10
11
4
10
3
NIGEB-195
3
10
5
4
2
NIGEB-90
3
9
13
14
3
2
NIGEB-44
9
10
5
11
2
NIGEB-113
7
8
15
2
NIGEB-78
5
7
15
6
7
5
NIGEB-74
6
7
15
4
NIGEB-187
10
9
27
8
10
1
1
NIGEB-183
11
6
13
10
5
8
NIGEB-97
10
11
7
6
4
2
NIGEB-135
6
9
18
10
NIGEB-101
10
10
20
10
7
4
NIGEB-107
4
7
16
4
13
4
4
NIGEB-84
10
6
15
4
8
4
NIGEB-119
4
8
12
5
5
NIGEB-131
11
7
20
15
13
4
3
NIGEB-104
2
11
4
2
NIGEB-9171
3
5
12
3
4
2
3
NIGEB-9179
2
5
14
5
4
2
NIGEB-2525
4
5
14
3
10
6
NIGEB-31
8
9
10
3
9
NIGEB-56
3
5
14
3
5
4
2
NIGEB-66
5
6
12
5
10
2
2
NIGEB-45
7
5
14
5
12
4
2
NIGEB-K56
9
5
10
9
5
2
NIGEB-261R2
9
5
10
9
5
4
NIGEB-288
8
9
16
10
14
3
1
NIGEB-92
12
7
15
11
10
4
2
NIGEB-151
7
6
13
5
7
4
4
NIGEB-233R2
9
14
20
12
16
9
10
NIGEB-155
22
10
5
5
6
2
4
NIGEB-242R2
3
11
12
5
9
5
NIGEB-38
3
4
11
4
10
2
4
NIGEB-41
5
12
3
9
4
NIGEB-761
3
7
14
6
10
NIGEB-108
3
8
10
3
10
6
NIGEB-58
5
4
14
3
6
NIGEB-26
7
5
10
11
4
8
3
NIGEB-265
1)
Abbreviation of mentioned antimicrobial agents are AMP, ampicillin; KAN, kanamycin; CHL,
chloramphenicol; PEN, penicillin; STR, streptomycin; CRO, Cronobacteriocin DGH2 ; ENT,
Enterobacteriocin DGH4.
2)
Halo diameter in scale of mm.
166
167 Gholami et al., Comparison of antibiotics and bacteriocins antibacterial activity on …
Bacteriocin temperature resistance test
Considering that protein activity is
reduced in the high-temperature;
thermal diagnostic test for bacterial
supernatant in the range of different
temperatures was performed. Thus,
after the bacterial supernatant obtained
the amount of 50µL into single vials;
each vial was placed in a hotplate with
temperatures of 35, 45, 55, 75 and
100ºC for 30 minutes. 10 µL of each
vial was transferred on antibiogram
disks and disks placed on the plates that
were inoculated with pathogenic
bacteria and was incubated at 32ºC,
then formation of halo was investigated
after 24 hours. All samples were
readjusted to pH 6.0 with sterile 1.0 M
HCl or 1.0 M NaOH. In this
experiment,
unheated
supernatant
served as the control (Mathieu et al.,
1994; Gao et al., 2010).
Bacteriocins and antibiotics sensitivity
determination
Bacterial strains of Xanthomonas were
tested
for
sensitivity
to
the
Cronobacteriocin
DGH2
and
Enterobacteriocin DGH4, and to some
antibiotics
such
as;
ampicillin,
kanamycin,
chloramphenicol,
penicillin, and streptomycin. Initial
testing was performed by the DD AWDA1 method as described by
Gholami
et
al.
(2013).
For
Xanthomonas
strains,
minimal
inhibitory concentrations (MICs) were
determined by the broth micro-dilution
1
Disc Diffusion – Agar Well Diffusion Assay
method in 96-well microtiter plates
(Becton Dickinson Labware, Franklin
Lakes, NJ) as described by the Clinical
and Laboratory Standards Institute
(CLSI) 2009. Mueller-Hinton Broth
from Germany (Merck Company),
ampicillin,
kanamycin,
chloramphenicol,
penicillin,
and
streptomycin from USA (Sigma
Company) were used in this
experiment.
Preparation of stock dilution and broth
dilution
The stock solutions are created in
higher concentrations to preserve their
qualities and afterward stored in
aliquots at -20°C. Dilution range of
some antibacterial that is needed for
this research is shown in Table 2. Stock
dilutions were prepared of the antibiotic
in 1000 and 100 µg L-1 concentrations
as required from primary stock solution
(10,000 mg L-1). A minimum of 1 mL
of each dilution per vial is needed for
the experiment. The antibacterial agent
(antibiotic or bacteriocin) is diluted in
sterile broth followed by mixed with
broth inoculated with bacterial strains.
Preparation of broth microdilution
100 μL of antibacterial agent dilution
are distributed into the microwell plates
then inoculated with 5 μL of overnight
broth of the test organism which were
diluted to 1 in 1000 in an appropriate
broth culture. The amount of the
inoculums affected the outcome of the
test significantly.
Iranian Journal of Fisheries Sciences 17(1) 2018
168
Table 2: Suggested antibiotic microdilution ranges for MIC and MBC Testing.
Antimicrobial agent
Concentration range (ug mL-1)
0.015 – 32
Penicillin G*
0.15 – 32
Ampicillin*
0.06 – 256
Chloramphenicol*
0.06 – 256
Tetracycline*
16 – 256
Streptomycin*
14 – 2500
Sakacin DGH6
0.15 – 32
Ciprofloxacin
*Source- (Working Party on Antibiotic Sensitivity Testing of the British
Society for Antimicrobial Chemotherapy, 1991).
Microwell plates were incubated for 18
hours at 37ºC and 500 μL of broth
culture inoculated with the organism
and kept at +4ºC in a cold room
overnight to be used as standard.
Determination of MIC by the broth
micro dilution method
The Broth micro dilution procedure is
one of the easiest and most convenient
methods for assessment of a small
number of isolates. (Domig et al., 2007;
Kushiro et al., 2009). This method
employs double-strength Mueller-Hinton
broth and 4X strength antibiotic solutions
prepared as serial two-fold dilutions.
Also the test organism is at a
concentration of 0.5 McFarland. A
mixture of double-strength MuellerHinton broth (100 mL), the antibiotic
dilutions (50 mL) and the target
organism suspension were incubated for
18-24 h at 35ºC. The MIC of the
antibacterial agents will be considered
the lowest concentration showing
inhibition of growth (Pfaller et al.,
2010).
Determination of MBC by the broth
microdilution method
The ‘Broth microdilution’ method
which was used in MIC determination
can willingly be converted to determine
the MBC as well (Bär et al., 2009;
Mandal et al., 2010).
Reading of results
MIC is explicit as the minimum
concentration of antibacterial agent
inhibited growth arbitrage by lack of
turbidity in the test tube. Standard strain
of recognized MIC is applied to check
the antibacterial activity and statements
as the control.
Assay of activity and determination of
antimicrobial spectrum
Antimicrobial substance (bacteriocin)
produced by the strain was detected by
DD - AWDA method. For DD AWDA method, Nutrient agar plates
(1.5% agar) were overlaid with 5 ml of
soft agar (0.75% agar) inoculated with
100µL of an overnight culture of the
indicator
microorganism.
The
concentration of bacteria in the
suspension was adjusted to 1.5 ×108
CFU/mL using Mc Farland’s scale (0.5
McFarland) (Patel et al., 2011; Nazemi
169 Gholami et al., Comparison of antibiotics and bacteriocins antibacterial activity on …
et al., 2017) and confirmed by a serial
dilution plating method (Blodgett,
2005). 15µL of bacteriocin was placed
in each disc and disc was moved on the
top agar layer that had hardened. The
plates were incubated for 24 h at 35ºC
and afterwards, zones of inhibition were
measured.
Antibacterial activity of bacteriocins
and antibiotics
Antibacterial effects of five types of
antibiotics
including
ampicillin,
kanamycin,
chloramphenicol,
penicillin,
streptomycin,
and
Cronobacteriocin
DGH2
and
Enterobacteriocin
DGH4
were
evaluated
on
107
strains
of
Xanthomonas (Jayapradha et al., 2009)
(Table 1).
Results
Bacteriocin
enzymatic
digestion
identification test
As shown in Table 3, after treatment by
proteolytic enzymes such as proteinaseK, trypsin and pepsin the antibacterial
activity of secreted components was
decreased by bacterial strains, but the
inhibitory activity of cell free
supernatant was not significantly
diminished
when
treated
with
papainase. Catalase had no effect on the
antimicrobial activity indicating that the
inhibitory activity is not due to
hydrogen peroxide production (results
not shown). This result showed that
these components are proteinaceous
molecules, and probably bacteriocins
(Todorov et al., 2011a; Jiang et al.,
2012).
Bacteriocin temperature resistance
identification test
Halo formation of the bacterial
supernatant in the temperature range of
25, 45, 65, and 100°C was investigated.
The zone formation at 25 and 45°C did
not change but reduced at 65°C and
there wasn't any zone at 120°C (Table
3). It can be deduced from this
experiment that the antibacterial
activity is factor that has a protein
structure (Garver and Muriana, 1993;
Todorov et al., 2011b).
Bacterial susceptibility
Table 2 shows MIC data for antibiotics
and bacteriocins against Xanthomonas
strains. Based on this analysis,
Xanthomonas
strains
were
comparatively susceptible and resistant
to
Cronobacteriocin
DGH2,
Enterobacteriocin DGH4, ampicillin,
kanamycin, chloramphenicol, penicillin
and streptomycin. MBC data for these
antimicrobial agents also was shown in
Table 4.
Iranian Journal of Fisheries Sciences 17(1) 2018
170
Table 3: Stability of bacteriocins produced by Cronobacter sp. DGH1 and Enterobacter sp. DGH3
after treatment with enzymes, detergents, urea, heat, NaCl and pH.
Enterobacteriocin Cronobacterioci
Effector
Concentration
DGH4*
n DGH2*
Proteinase K
1mg mL-1
_
Pepsin
1mg mL-1
_
Trypsin
1mg mL-1
_
pH 3-6
3h
+
++
pH 6-8
3h
++
++
pH 8-10
3h
+
+
Temperature 4, 55, 60, 85, 100 ºC
1h
+
+
Temperature 25, 35,45 ºC
1h
+
++
Temperature 121 ºC
20 min
_
Triton X-100, Tween 20, Tween 80
1% (v/v)
+
++
SDS, urea, NaCl, EDTA,
1% (w/v)
+
++
manganese sulphate
1% (w/v)
++
++
*Activity of bacteriocin was expressed in + = presence of inhibition zone (1-5 mm); ++ = presence of
inhibition zone (5-16mm); − = no inhibition.
Table 4: Information about antimicrobial agents, break point, MIC and MBC range and
antimicrobial resistance percentage for 107 samples of Xanthomonas citri subsp.citri.
No. of isolates resistant
to antimicrobial agents
Antimicrobial
(%)
1
agent
Xanthomonas strains
(n=107)
≥32
<184 - >390
<368 - >780
5.6
AMP
≥16
<115 - >340
<225 - >899
3.7
KAN
≥32
<90 - >257
<115 - >350
0.9
CHL
≥4
<315- >610
<315- >610
13.1
PEN
≥64
<210 - >512
<504 - >1536
3.7
STR
≥4
<400- >2048
<550- >3000
35.5
CRO
≥4/76
<182 - >390
<370 - >782
41.1
ENT
No. of multi-drug resistant (MDR) isolates 3
2.8
1)
Abbreviation of mentioned antibiotics are AMP, ampicillin; KAN, kanamycin; CHL, chloramphenicol;
PEN, penicillin; STR, streptomycin; CRO, Cronobacteriocin DGH2; ENT, Enterobacteriocin DGH4
2)
Breakpoints were adopted from CLSI (Clinical and Laboratory Standards Institute)
3)
In this study isolates which were resistant to at least 4 groups of antimicrobial agents considered as
MDR Xanthomonas citri subsp.citri strains
Breakpoint for
resistance (µg mL1 2
)
MIC range in
isolates (µg mL1
)
Bacterial resistance
Among the antimicrobials, most of
Xanthomonas strains are resistant to
Enterobacteriocin DGH4 (41.1%);
While the lowest of strains are resistant
to chloramphenicol (0.9%). Since the
bacteriocin functional range is much
smaller than antibiotics. Such results
were quite predictable. Among types of
antibiotics the most common bacterial
MBC range in
isolates (µg
mL-1)
strains resistant to the penicillin were
shown. Penicillin eliminates grampositive bacteria and Xanthomonas is
the gram-negative bacterium, therefore
most bacterial strains studied were
resistant to penicillin. The inhibitory
effect of this antibiotic is lower than
other antibiotics (Table 4). MIC range
of the isolates indicated that strains
show the greatest sensitivity to
171 Gholami et al., Comparison of antibiotics and bacteriocins antibacterial activity on …
chloramphenicol and have the least
sensitivity to the Enterobacteriocin
DGH4, on the other hand; MBC range
of this strain also gives similar results
(Table 4). As can be seen in Table 2,
number of multi drug resistance (MDR)
strains (Kazem et al., 2012) are
included in 2.8% of the total
monocytogenes.
Comparison
of
sensitivity
and
resistance to antibiotics
As can be seen in Table 1,
chloramphenicol has the most potent
inhibitory effect among antibacterial
agents. NIGEB-183 strain is the most
sensitive to these antibiotics by formed
a halo with 27 mm diameter. Only
NIGEB-242R1strain is resistant to
chloramphenicol. Penicillin also has
minimum
inhibitory
effects
on
Xanthomonas strains, and strains of
NIGEB-142, NIGEB-136, NIGEB-96,
NIGEB-180, NIGEB-65, NIGEB-1842,
NIGEB-116,
NIGEB-263R1,
NIGEB-90, NIGEB-44, NIGEB-78,
NIGEB-101, NIGEB-9171 and NIGEB2525 are resistant to this antibiotic, but
strain of NIGEB-104 is the most
sensitive to this antibiotic with a 27 mm
zone diameter. Strains of NIGEB-142,
NIGEB-761, NIGEB-90, NIGEB-761,
NIGEB-90, NIGEB-113, NIGEB-9171
and NIGEB-184-2 are resistant to
ampicillin while the strain of NIGEB242R2 is the most sensitive to this
antibiotic with a 22 mm zone diameter.
Furthermore, strains of NIGEB-170,
NIGEB-142, NIGEB-211R1-1 and
NIGEB-116 are resistant to kanamycin
while strain of NIGEB-79 is most
sensitive to this antibiotic with a 16 mm
zone diameter. Strains of NIGEB-187,
NIGEB-184-2,
NIGEB-116
and
NIGEB-112
are
resistant
to
streptomycin whereas NIGEB-155
strain is most sensitive to streptomycin
with a 16 mm zone diameter. One
example of antibiotic antibacterial
activity was shown in Fig. 1.
Comparison
of
sensitivity
and
resistance of Xanthomonas strains to
bacteriocins
Thirty eight strains are resistant to
Cronobacteriocin sp. DGH2; NIGEBK32 strain is the most sensitive to this
bacteriocin with an 11 mm zone
diameter. Forty seven strains are
resistant to Enterobacteriocin DGH4
whereas NIGEB-155 strain is the most
sensitive to this bacteriocin with a 10
mm zone diameter (Table 1). One
example of bacteriocin antibacterial
activity was shown in Fig. 1.
Iranian Journal of Fisheries Sciences 17(1) 2018
172
Figure 1: Antibacterial activity of antimicrobial agents that were examined in this study. a:
ampicillin, b: penicillin, c: Enterobacteriocin DGH4, d: chloramphenicol, e:
Cronobacteriocin DGH2, f: kanamycin, g: streptomycin, n: negative control. Plate
surface inoculated with pathogenic bacteria (NIGEB-65 strain).
Discussion
Citrus is one of the most universal fruit
crops with high economic and health
value and is important in daily life
(Pergola et al., 2013). Citrus fruit is
produced all over the tropic and
subtropical areas of the world, where
there is sufficient water, rich soil and
adequate moisture for tree growth and
fruit production. These natural products
in addition to their use as a food source
are also an important component with
many pharmaceutical and healthcare
applications (Gmitter and Hu, 1990).
The pathogen Xanthomonas citri
subsp. Citri is a Gram-negative
bacterium which is the main factor of
citrus canker, a threatening disorder that
affected citrus crops all around the
world (Brunings and Gabriel, 2003;
Paola et al., 2011). Antibiotics were
very important discovery in the healthy
life of plants and animals, since the
recognition of penicillin in 1928 as the
first antibiotic, and come behined by it
full-scale output (Bennett, 2001). Babes
and Cornil found an antagonism in
Staphylococcus genus in 1885 (Cornil
et al., 1885). Gratia first found
bacteriocins in 1925 (Gratia, 1925). All
these discoveries are confined to a few
years and it is exposition the
importance of bacteriocins. As the
functional limited area of antibiotics is
more than bacteriocins (Kheadr et al.,
2004; Pogány et al., 2010; Naghmouchi
et al., 2012), in this study, we attained a
resembling conclusion and inhibitory
effects of antibiotics on Xanthomonas
strains is a wider span of bacteriocins,
whereas activity of Xanthomonas
strains was controlled by each
bacteriocin was less than types of
antibiotics. In this project the range of
concentration of each antibiotic is from
256 μg mL-1 to 0.015 μg mL-1. This
range for bacteriocins is 14 to 2500 μg
mL-1. Studies have shown that the
antibacterial effects of antibiotics on
pathogenic bacteria have begun three
173 Gholami et al., Comparison of antibiotics and bacteriocins antibacterial activity on …
years ago and have contributed in the
inhibition of microbial diseases and
infections (Hayes G Fau-Moens et al.,
2013). Comparison of antibacterial
effects and the range of antibiotics with
bacteriocins has not been studied well
(Kheadr et al., 2004). On the other
hand, comparison of antibacterial
effects and the range of antibiotics with
bacteriocins inhibiting the bacterial
citrus canker disease has been
examined for the first time in this
project and no previous studies have
been done in this context and on
Xanthomonas pathogenic bacterium
strains, so far. Considering that the best
way to control citrus canker disease is
the use of antibiotics, and pathogenic
strains are sensitive to these antibiotics,
in order to better control the disease by
antibiotics, examination of the rate of
bacterial
resistance
to
these
antimicrobial agents is useful. Knowing
that these strains were inhibited by
either antibiotic or how much
antibiotics is needed to control bacteria
is cost-effective economically, and
saves antibiotics application. As
mentioned above, most of the inhibitory
effect among antibacterial agents is
chloramphenicol, NIGEB-183 strain is
the most sensitive to these antibiotics,
and only NIGEB-242R1strain is
resistant to chloramphenicol. Because
Penicillin is an antibiotic inhibitor of
Gram-positive bacteria, it was expected
that it has a small anti-bacterial effect
on the Xanthomonas strains as a Gramnegative bacteria. Bacteriocins have a
narrow antibacterial spectrum to
antibiotics; for this reason, strains that
were resistant to bacteriocins were
more. The study shows that the best
antibiotics to inhibit strains of the
pathogenic
bacteria,
is
chloramphenicol. The required amount
of this antibiotic to inhibit bacteria is
less than other antibacterial agents.
Moreover, the range of its antibacterial
activity is greater than other
antimicrobial agents.
Acknowledgements
We appreciate the National Institute of
Genetic Engineering and Biotechnology
(NIGEB) for providing the research
facility.
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