Molecular Characterization of Some Bacteria Isolated from Broiler Chickens Showing Respiratory Manifestations

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98
Alexandria Journal of Veterinary Sciences
www.alexjvs.com
AJVS. Vol. 55 (2): 98-106. Oct. 2017
DOI: 10.5455/ajvs.249636
Molecular Characterization of Some Bacteria Isolated from Broiler Chickens
Showing Respiratory Manifestations
Eman M.M. Elghazaly1, Eman k. Sedeek1, Samy A. Khalil2
1Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Matrouh branch
2Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University
ABSTRACT
Key words:
E. coli, P. aeru-
ginosa, virulence
genes, antibiogram,
plasmid profile
Fermented wheat germ extract (FWGE) is a multisubstance composition contains 2-
methoxy benzoquinone and 2, 6-dimethoxy benzoquinone which are likely to exert some of
its biological effects as well as it is a concentrated source of vitamins, minerals, and protein.
An experimental trial of FWGE supplementation to broiler feed from one day old with a
rate of 0.5, 1.5 and 3 g/kg feed was tried. Results revealed that all doses of FWGE increased
body weight significantly (p≤0.05), especially with the dose of 3 g/kg feed. Also, FCR
values decreased significantly (p≤0.05) in the FWGE treated groups. Regarding biochemical
analysis at 35 days old, the most significant results obtained with the doses of 1.5 and 3 g
FWGE/kg feed especially in SGOT, SGPT, creatinine, uric acid, total protein, glucose, and
triglycerides levels. Physiologically, FWGE only increased hemoglobin concentration
significantly (p≤0.05) without alteration of red blood and white blood cells counts. There
was also a significant increase (p≤0.05) in the intestinal weight in relation to carcass
weight% and a significant decrease (p≤0.05) in the liver and total body fat weights in
relation to carcass weight%. At 45 days (10 days after vNDV challenge), the mortality rates
were 60% in the non-treated non-vaccinated challenged chicken group 5 and 4% in the non-
treated vaccinated challenged chicken group 4 without appearance of any mortality in the 3
FWGE treated groups. Also, addition of FWGE had a positive effect on HI titers for NDV
in the collected serum samples at 45 days old. Finally, it was concluded that FWGE
improved the general health condition of broilers regarding biochemical and physiological
parameters and immune response to NDV vaccination.
Correspondence
to:
emanmoneer1@gmail.c
om
1. INTRODUCTION
Diseases of the respiratory tract have an
important role in poultry. Among the systemic
disease, respiratory system diseases generally
ranked first (Glisson, 1998). A mixed infection is
the main causative agent of chronic respiratory
diseases in chickens (Nunoya et al., 1999).These
diseases may result as either primary or multi
system diseases. Respiratory system diseases may
result from bacteria, virus, parasite, fungi or
nutritional environmental factors (Glisson, 1998)
Escherichia coli is considered one of the
most serious bacteria that affecting poultry and
other animals and causing significant economic
losses in poultry industry and also affecting humans
(Moulin scholeur et al., 2007). E. coli has an
important role in lower-respiratory-tract infections
in poultry. Although in severe cases mortality can
be over 20%. It is associated with high morbidity
and loss in productivity resulting in greatest
economic loss (Gross, 1994).
Temperature-sensitive haemagglutinin (tsh)
of E. coli plays a significant role in the colonization
of air sacs (Dozois et al., 2000); P-fimbriae (pap)
are responsible for the adhesion to internal organs in
later stage (Dho-Moulin and Fairbrother, 1999).
Pseudomonas aeruginosa primarily affects the
upper respiratory tract of poultry, causing rhinitis,
sinusitis and laryngitis (Bailey et al.,
2000).Pathogenicity of P. aeruginosa in birds is
mainly associated with septicemic and respiratory
infections and sinusitis (Hai-ping, 2009).
P. aeruginosa are able to survive in variable
environmental conditions due to its versatile
nutritional abilities and its ability to resist high
concentrations of common antibiotics (Aumeran et

Elghazaly et al. 2017. AJVS 55(2): 98-106
99
al., 2007). Resistance to antibiotic has been occurred
due to combination of restriction of antibiotic
uptake through the outer membrane and a variety of
energy-dependent mechanisms (Presteri et al.,
2007). P. aeruginosa is a classic opportunistic
pathogen because it has the ability to resist many
antibiotics and disinfectants; and also due to it has a
large number of putative virulence factors and
acquired resistance due to plasmids (Shahid and
Malik, 2004).
Staphylococcus aureus is considered to be a
normal resident of the chicken, located on the skin
and feathers and in the respiratory and intestinal
tracts. Avian Salmonellosis caused by Salmonella
species is related to economic problems that occur
in all stages of poultry industry from production to
marketing. It resulted in drop of egg production,
fertility, hatchability and increased early chick
mortality (Abd - Ellatef, 1995).
The aim of the study was planned for
isolation and identification of bacteria from broiler
chickens showing respiratory manifestations,
Confirmation of biochemically identified E. coli
isolates by PCR as an accurate and sensitive
technique, detection of virulence genes of E. coli
associated with respiratory system infection,
detection of sensitivity of P. aeruginosa to different
antibacterial agents and Plasmid profile of P.
aeruginosa.
2. MATERIALS AND METHODS:
2.1. Sampling:
A total of 41samples (16 nasal swabs, 11tracheal
swabs and14lung samples) were collected. The
collected swabs were immersed into nutrient or
trypticase soya broth and transferred to the
laboratory of Microbiology Department, Faculty of
Veterinary Medicine, Alexandria University in an
insulated ice box without delay to be subjected for
bacteriological examination.
2.2. Isolation and identification of bacteria from
broiler chickens with respiratory manifestations:
The collected samples were immersed into tryptic
soya broth or nutrient broth and incubated at 37 ˚C
for 18hrs then subcultured into 5% blood agar base,
nutrient agar, Mannitol salt agar, MacConkey`s
agar, Eosin methylene blue agar medium, Cetrimide
agar medium and Muller-Hinton agar medium and
incubated at 37 ˚C for 24-48hrs.suspected colony
from different media were picked up and subjected
to morphological and biochemical identification
(Quinn et al., 2011).
2.3. Antibacterial sensitivity test for isolated P.
aeruginosa:
After preparation of McFarland's No.1 from
P. aeruginosa isolates, a sterile Pasteur pipette was
used to inoculate the suspension on the surface of
Muller Hinton agar plate. Excess fluid was removed
by the pipette then the plate was incubated for about
30 min. The chosen antibiotics discs were applied to
adequate spacing so that two discs shouldn`t closer
than 24 mm from one center to the other center and
no more than 15 mm from the edges of Petri dish by
using sterile fine pointed forceps. The discs were
pressed gently to ensure full contact of discs to the
medium, and then plates were incubated at 37 ˚C for
24 hours. After incubation, the degree of sensitivity
was determined by measuring the easily visible and
clear zone of inhibition of growth produced by
diffusion of antibiotics from disc into the
surrounding medium. The results were interpreted
according to NCCLS (1990).
2.4. Plasmid profile of isolated P. aeruginosa:
The isolated and biochemically identified P.
aeruginosa isolates were tested for presence or
absence of plasmid using alkaline lysis method
(Sambrook et al., 1989). For detection of plasmid
DNA expression after Subculture (five times) of P.
aeruginosa in presence of half Minimum Inhibitory
Concentration (MIC) of ofloxacin antibiotic; one
disc of ofloxacin was dissolved (200 mg / L) in
falcon tube containing 10 ml distilled water. About,
25 μl from antibiotic suspension containing 0.5 MIC
were added to one litter of sterile nutrient agar
medium after its cooling to 50˚C to avoid
destruction of antibiotic. The medium containing
half MIC of ofloxacin antibiotic (0.5 mg /L) was
poured into sterile Petri dishes and left to dry.
Bacterial isolates having plasmid DNA were
streaked into nutrient agar medium containing half
MIC of ofloxacin antibiotic. Plates were incubated
at 37˚C for 24hrs. The previous steps were repeated
five times to detect effect of half MIC of ofloxacin
antibiotic (0.5 mg /L) on expression of plasmid
DNA by alkaline lysis method (Sambrook et al.,
1989). For detection of plasmid DNA expression
after Subculture of P. aeruginosa in absence of
antibiotic; a sterile nutrient agar medium without
antibiotic was poured into sterile Petri dishes and
left to dry. Bacterial isolates having plasmid DNA
were streaked into nutrient agar medium without
antibiotic. The plates were incubated at 37˚C for 24
hrs. The previous steps were repeated (5, 10 and 15
times) to detect effect of subculture of bacteria
containing plasmid DNA in absence of antibiotic on
expression of plasmid DNA by alkaline lysis
method (Sambrook et al., 1989).
2.5. Molecular identification of isolated E. coli:
a. DNA extraction:

Elghazaly et al. 2017. AJVS 55(2): 98-106
100
DNA Extraction was carried out for 21
biochemically identified E. coli isolates by boiling
method (Sambrook and Russell, 2001).
B. DNA Molecular weight marker:
The ladder was mixed gently by pipetting up and
down. Six μl of the required ladder were directly
loaded.
C. Agarose gel electrophoresis (Sambrook and
Russell, 2001).
1. RESULTS AND DISCUSSION
In recent years respiratory diseases become
the main hazards to the poultry industry causing
significant economic losses. When chicks are
exposed to stress, bacteria often penetrate protective
barriers of the respiratory tract and causing severe
damage to the heart and lungs by causing chronic
respiratory disease (Lin et al., 1993), so the aim of
this study to identify bacteria causing respiratory
manifestations in broiler chickens.
In this study the results of identification of bacteria
isolated from respiratory system of broiler chickens
suffering from respiratory manifestations revealed
that the most commonly isolated bacteria were E.
coli, P. aeruginosa, Corynebacterium spp,
Coagulase negative Staphylococcus, Salmonella
Entritidis, Staphylococcus aureus and Pasteurella
multocida which were isolated at a percentage of
33.3%, 26.9%, 15.9%, 11.1%, 6.4%, 4.8% and
1.6%, respectively as shown in table (4). The
obtained results were similar to that reported by
Mamza et al. (2010) who isolated E. coli from lung
(15.5%) and trachea (15.3%) at a total percentage of
30.3%. these results were lower than that reported
by Murthy et al. (2008) who isolated E. coli and
Pasteurella multocida at a percentage of 51.9% and
9.6% , respectively.
Table (1): Primer sequences for amplification of virulence genes of E. coli.
Table (2): Components of PCR reaction used for detection of virulence genes of E. coli
Table (3): Cycling conditions of the primers used for amplification of genes of virulence of E. coli
Gene
Initial
denaturation
Denaturation
Annealing
Extension
No. of
cycles
Final extension
Papc
94˚C
5 min.
94˚C
1min.
63˚C
1min.
72˚C
2 min.
30
72˚C
10 min.
Tsh
94˚C
5 min.
94˚C
1min.
55˚C
1min.
72˚C
2 min.
30
72˚C
10 min.
PhoA
94˚C
5 min.
94˚C
30 sec.
58˚C
45 sec.
72˚C
45 sec.
35
72˚C
10 min.
Fimh
95˚C
2 min.
94˚C
30 sec.
58˚C
30 sec.
72˚C
1min.
33
72˚C
7 min.
Table (4): Bacteria isolated from broiler chickens suffering from respiratory manifestations.
Isolated bacteria
No. of isolates
%
Reference
Amplified
Product (bp)
Primer sequence (5'-3')
Primer used
Target gene
Hu et al., 2011
720
CGATTCTGGAAATGGCAAAAG
phoA-F
PhoA
CGTGATCAGCGGTGACTATGAC
phoA-R
Tiba et al., 2008
328
GACGGCTGTACTGCAGGGTGTGGCG
Papc-F
Papc
ATATCCTTTCTGCAGGGATGCAATA
papc-R
Provence and curtiss,
1994
620
GGTGGTGCACTGGAGTGG
TSH-F
TSH
AGTCCAGCGTGATAGTGG
TSH-R
Tiba et al., 2008
508
TGCAGAACGGATAAGCCGTGG
fimH-f
FimH
GCAGTCACCTGCCCTCCGGTA
FimH–R
Component
Volume/reaction
Emerald Amp GT PCR master mix (2x premix)
12.5μl
Forward primer(20 pmol)
1.25μl
Reverse primer (20 pmol)
1.25μl
Template DNA
10μl
Total
25 μl

Elghazaly et al. 2017. AJVS 55(2): 98-106
101
E. coli
21
33.3
P. aeruginosa
17
26.9
Salmonella enteritidis
4
6.4
Pasteurella multocida
1
1.6
Staphylococcus aureus
3
4.8
Coagulase negative Staphylococcus
7
11.1
Corynebacterium species
10
15.9
Total
63
100
%: according to total number of isolated bacteria
Table (5): Incidence of bacteria isolated as a single culture from broiler chickens suffering from respiratory manifestations:
No. of examined samples = 41 % was calculated according to total number of samples.
Table (6): Incidence of bacteria isolated as mixed culture from broiler chickens suffering from respiratory
manifestations:
Suspected isolates
NO. of mixed
culture
%
E. coli and Corynebacterium species
1
2.4%
E. coli and Staphylococcus aureus
1
2.4%
E. coli and Salmonella Enteritidis
1
2.4%
E. coli and P. aeruginosa
2
4.9%
P. aeruginosa and Corynebacterium species
1
2.4%
Salmonella Enteritidis and Staphylococcus aureus
1
2.4%
Pasteurella multocida and Corynebacterium species
1
2.4%
Coagulase negative Staphylococcus and Corynebacterium species
2
4.9%
Total
10
24.4%
No. of examined samples = 41 % was calculated according to total number of samples
This disagreement may be due to samples
collected under complete aseptic condition which
reduce the chance for environmental bacteria to
grow. These results were higher than that reported
by Berag and Elhassan (1987) who isolated E. coli
and P. aeruginosa at a percentage of 17.92% and
10.4%, respectively and this dis agreement may be
due to using different breed, age or season.
Results of amplification of E. coli (phoA)
coding gene by using PCR. Twenty one isolates of
biochemically identified E. coli were randomly
studied for detection of phoA gene using PCR
technique. The specificity of the primers was
confirmed by positive amplification of fragment
with the extracted DNA of the bacterial isolate. Out
of 21 tested isolates, ten isolates (47.6%) were
positive for the phoA gene as shown in figure (1, 2,
and 3). The PCR assay yielded amplified products
of 720bp specific for (phoA) gene.
Results of amplification of E. coli virulence
genes associated with respiratory system by PCR
(tsH, fimH and papC): out of ten detected E. coli
isolates by PCR, only 3(30%) E. coli isolates were
positive for tsh gene (specific band at 620bp) as
shown in figure (4), 7(70%) E. coli isolates were
positive for fimH gene (specific band at 508bp) as
shown in figure (5), only 1(10%) E. coli isolate was
positive for papc gene (specific band at 328bp) as
shown in figure (6).
Suspected isolates
NO. of single culture
%
E. coli
14
34.1%
P. aeruginosa
6
14.6%
Coagulase negative Staphylococcus
4
9.8%
Staphylococcus aureus
1
2.4%
Corynebacterium species
4
9.8%
Salmonella Enteritidis
2
4.9%
Pasteurella multocida
0
0%
Total
31
75.6%

Elghazaly et al. 2017. AJVS 55(2): 98-106
102
Figure (1): Agarose gel electrophoresis (1.5 %) of the amplified pho A gene of the isolated E. coli: Lane (1): DNA molecular
weight ladder (100bp ladder). Lanes (2, 3, 4 and 7) indicate positive results for phoA gene (specific band of 720bp). Lane (9): control
positive for phoA gene. Lanes (5, 6 and 8): are negative results for phoA gene.
Figure (2): Agarose gel electrophoresis (1.5 %) of the amplified phoA gene of the isolated E. coli. Lane (1): DNA molecular
weight ladder (100bp ladder). Lane (2): control positive for phoA gene. Lanes (3, 4 and 8): Positive results for phoA gene (specific
band of 720bp). Lanes (5, 6, 7 and 9): negative results for phoA gene.
Figure (3): Agarose gel electrophoresis (1.5 %) of the amplified phoA gene of the isolated E. coli. Lane (1): DNA molecular
weight ladder (100bp ladder). Lane (2): control positive for phoA gene. Lanes (5, 6 and 7): Positive results for phoA gene (specific
band of 720bp). Lanes (3, 4, 8 and 9): negative results for phoA gene.
Figure (4): Agarose gel electrophoresis (1.5 %) of the amplified tsh gene of the isolated E. coli. Lane (1): DNA molecular weight
ladder (100bp ladder). Lanes (5, 6and7): Positive results for tsh gene (specific band of 620bp). Lanes (2, 3, 4, 8, 9, 10 and 11):
negative results for tsh gene.
Figure (5): Agarose gel electrophoresis (1.5 %) of the amplified fim H gene of the isolated E. coli. Lane (1): DNA molecular weight ladder (100bp
ladder). Lanes (2, 5, 6, 7, 8, 10 and 11): Positive results for fimH gene (specific band of 508bp). Lane (3, 4and 9): negative results of fim H gene.
.
Figure (6): Agarose gel electrophoresis (1.5 %) of the amplified papC gene of the isolated E. coli. Lane (1): DNA molecular
weight ladder (100bp ladders). Lane (10): Positive results for papc gene (specific band of 328bp). Lanes (2, 3, 4, 5, 6, 7, 8, 9 and 11):
negative results for papC gene.

Elghazaly et al. 2017. AJVS 55(2): 98-106
103
Table (7): Results of antibiotic sensitivity of P. aeruginosa and some E. coli isolates:
Antibiotics
No. of isolates
S
I
R
P.
aeruginosa
E.
coli
P.
aeruginosa
E. coli
P.
aeruginosa
E. coli
P.
aeruginosa
E. coli
No.
%
No.
%
No.
%
No.
%
No.
%
No.
%
Levofloxacin
17
3
1
5.9
1
33.3
0
0
0
0
16
94.1
2
66.7
Erythromycin
0
0
0
0
1
5.9
2
66.7
16
94.1
1
33.3
Streptomycin
1
5.9
1
33.3
0
0
0
0
16
94.1
2
66.7
Norfloxacin
1
5.9
1
33.3
3
17.7
0
0
13
76.5
2
66.7
Rifampin
0
0
0
0
0
0
0
0
17
100
3
100
Chloramphenicol
0
0
0
0
1
5.9
1
33.3
16
94.1
2
66.7
Neomycin
0
0
0
0
1
5.9
2
66.7
16
94.1
1
33.3
Ciprofloxacin
1
5.9
1
33.3
0
0
0
0
16
94.1
2
66.7
Ofloxacin
1
5.9
1
33.3
0
0
0
0
16
94.1
2
66.7
%: according to number of isolates, R: resistant, S: susceptible, I: intermediate susceptible.
The obtained results showed that presence of phoA
gene in E. coli isolated from broiler chickens with
respiratory manifestations at a percentage of 47.6%
that higher than the result obtained by (Rasha et
al.,2015) who recorded that PCR for amplification
of phoA gene of E. coli at a percentage of 37.5%.
This disagreement may be due to difference in the
site of isolation from broiler chickens.
The recovered results showed that presence
of tsh in E. coli isolated from broiler chickens with
respiratory manifestations at a percentage of 30%
that agree with the result reported by Oh et al.,
(2011) who found that PCR for amplification of tsh
gene of E. coli at a percentage of34.48% and lower
than the result obtained by (Roussan et al., 2014)
who identified tsh gene with a percentage (66%) by
using PCR.
This study detected presence of fimH gene
in E. coli isolated from respiratory system of broiler
chicken at a percentage of 70%. This result is higher
than the result that said by(Mbanga and Nyararai,
2015) who said that percent of PCR for
amplification of fimH gene of E. coli was (33.3%)
of APEC isolates and lower than the result obtained
by (Roussan et al., 2014) who identified fimH gene
with a percentage (94%) by using PCR.
In this study, the percentage of papC gene
was 10% which is lower than the result obtained by
(Rocha et al., 2008) who found that papC gene with
percentage of 24.3%.
As shown in table (7), the antibiotic
resistance pattern showed that P. aeruginosa
showed high resistance to levofloxacin (94.1%),
streptomycin (94.1%), ciprofloxacin (94.1%),
ofloxacin (94.1%), erythromycin (94.1%),
chloramphenicol (94.1%), neomycin (94.1%) and
norfloxacin (76.5%) but intermediate susceptible to
erythromycin (5.9%), chloramphenicol (5.9%) and
neomycin (5.9%) and norfloxacin (17.7 %) and
susceptible to levofloxacin (5.9%), streptomycin
(5.9%), ciprofloxacin (5.9%), ofloxacin (5.9%) and
norfloxacin (5.9%). This result agreed with that
conducted by (Olayinka et al., 2009) who reported
that P. aeruginosa showed resistance to
ciprofloxacin and chloramphenicol at a percentage
of 90.2% and 97.8%, respectively but higher than
that reported by (Akingbade et al., 2012) who found
that P. aeruginosa showed resistance to
erythromycin, streptomycin, ofloxacin,
ciprofloxacin at a percentage 72.7%, 65.5%, 60%
and 35.5%, respectively. This disagreement may be
due to difference in strains of bacteria. E. coli
isolates showed high resistance to rifampin (100%),
levofloxacin (66.7%), streptomycin(66.7%) ,
norfloxacin (66.7%), chloramphenicol (66.7%),
ciprofloxacin (66.7%), erythromycin (33.3%) and
neomycin (33.3%) but intermediate susceptible to
erythromycin (66.7%), neomycin (66.7%) and
chloramphenicol (33.3%) but susceptible to
levofloxacin (33.3%), streptomycin (33.3%),
norfloxacin (33.3%), ciprofloxacin (33.3%) and
ofloxacin (33.3%). This results agreed with that
revealed by (Moniri and dastehgoli, 2007) who
reported that E. coli showed resistance to
ciprofloxacin at a percentage of 69.7% but higher
than that reported by (Miles et al., 2006) who found
that E. coli showed resistance to ciprofloxacin,
norfloxacin and and ofloxacin at a percentage of
11.8%, 20.6% and 14.7%, respectively and this
higher resistance may be due to frequent using of
the same antibiotic in treatment which lead to
resistance to this antibiotic but lower than that
reported by (Jahantigh and Reza, 2015) who said
that E. coli isolates were resistant to ciprofloxacin
and norfloxacin at a percentage of 91% and 88%,
respectively.
Plasmid DNA profile of extracted plasmid
DNA from P. aeruginosa and some E. coli isolates:
Out of 17 P. aeruginosa isolates, 15(88, 2%)
isolates showed plasmid DNA profile (specific band

Elghazaly et al. 2017. AJVS 55(2): 98-106
104
at 25kbp) and out of 3 E. coli isolates, 2 (66.7%)
isolates also showed plasmid DNA profile at the
same size as shown in figure (7).
Results of plasmid DNA profile of plasmid
DNA extracted from P. aeruginosa and some E. coli
isolates after subculture with 0.5 minimal inhibitory
concentrations (MIC) of ofloxacin. All positive
plasmid DNA profile of P. aeruginosa isolates (15
isolates) and E. coli isolates (2 isolates) showed
plasmid DNA profile (specific band at 125kbp) after
subculture with 0.5 MIC of ofloxacin as shown in
figure (8).
Results of plasmid DNA profile of plasmid
DNA extracted from P. aeruginosa and some E. coli
isolates after (5, 10 and 15) subculture without
antibiotic: All positive plasmid DNA profile of P.
aeruginosa isolates (15 isolates) and E. coli isolates
(2 isolates) showed plasmid DNA profile (specific
band at 25kbp) after (5, 10 and 15) subculture
without antibiotics shown in figure (9, 10 and 11).
In this study plasmids were not disappear by (5,10
and15) subculture and this disagree with that
reported by Chadfield et al. (2001) who reported
that disadvantage of plasmid profiling are not
associated with epidemiological identical strains and
lost by repeated subculture of bacteria over long
period of times.
Figure (7): Agarose gel electrophoresis (1.5 %) of plasmid DNA extracted from P. aeruginosa and some E. coli isolates (specific band
at 25kbp)
Figure (8): Agarose gel electrophoresis (1.5 %) of plasmid DNA extracted from P. aeruginosa and some E. coli isolates (specific band
at 25kbp) after 5 subcultures with 0.5 MIC of ofloxacin.
Figure (9): Agarose gel electrophoresis (1.5 %) of plasmid DNA extracted from P. aeruginosa and some E. coli isolates (specific
band at 25kbp) after 5 subcultures without antibiotic.
Figure (10): Agarose gel electrophoresis (1.5 %) of plasmid DNA extracted from P. aeruginosa and some E. coli isolates (specific
band at 25kbp) after 10 subcultures without antibiotic.
Figure (11): Agarose gel electrophoresis (1.5 %) of plasmid DNA extracted from P. aeruginosa and some E. coli isolates (specific
band at 25kbp) after 15 subcultures without antibiotic.

Elghazaly et al. 2017. AJVS 55(2): 98-106
105
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