Genetic Linkage of the Antibiotic Resistance Ability in the Escherichia coli UR4 Strain Isolated from Urine
Sahar M. Ouda
It is of interest to understand the antibiotic resistance ability in pathogenic bacteria at the molecular level. Characterization of bacteria by methods based on phenotypic, biochemical and molecular characters were followed herein; and plasmids were certain target in the present study. Seventeen bacterial isolates were isolated from urine, blood and stool samples of patients with urinary tract infections, bloody diarrhea and fever. They were characterized by phenotypic and biochemical criteria and were identified as belonging to Escherichia coli (E. coli). One of them, isolate number 4 from urine (UR4) was resistant to 14 types of antibiotics used. Hence this strain was subjected to molecular identification. DNA was isolated from E. coli UR4 and 16SrRNA gene was separated after agarose gel electrophoresis and then was sequenced. The sequence was subjected to gene bank and showed about 99.5% similarity to E. coli category. Growing of E. coli UR4 at elevated temperature (42°C) and treatment of colonies with sodium dodycyl sulphate, sodium azide and ethidium bromide revealed a mutants lacking antibiotic resistance ability with mutation percentage ranging from 0.6-14%. The mutation in E. coli UR4 was a stable character and no recovery of mutants was observed. Plasmid profile of the E. coli UR4 wild strain and its four mutants showed five plasmids in the wild strain and four only in its mutants. One plasmid of a molecular mass of about 600 bp is showed to be deficient or dissociated indicating on its role in antibiotic resistance ability in the E. coli UR4 strain.
Received: January 28, 2013;
Accepted: March 15, 2013;
Published: May 21, 2013
Escherichia coli (E. coli) is a Gram-negative, rod-shaped, motile,
facultatively anaerobic and non-sporulating bacterium. It belongs to family
enterobacteriaceae in which most bacterial genera are human pathogens (Vogt
and Dippold, 2005). It is part of the microflora of human colon and can
benefit their hosts by producing vitamin K and by neutralization of pathogenic
bacteria within the intestine and can be changed to pathogenic bacteria under
certain conditions such as suppression of immune system (Reid
et al., 2001).
Transmission of E. coli is commonly by fecal/oral route with contaminated
foods. At least five types of intestinal infections that differ in pathogenic
mechanisms have been identified for E. coli viz., enterotoxigenic, enteropathogenic,
enterohaemorrhagic, enteroinvasive and enteroaggregative (Harvey
and Champe, 2012). E. coli was isolated from many pathogenic cases
such as gastroenteritis, neonatal meningitis, haemolytic-uremic syndrome, peritonitis,
mastitis, cystitis, pyelonephrities, urethrities, vaginities, endometrities
and prostatitis (Tauschek et al., 2002). Unfortunately,
many authors described the existence of E. coli variants resistant to
many antibiotics (Saffar et al., 2008). The antibiotic
resistance ability in E. coli is due to secretion of enzymes by target
strains, exopolyrsacharide production, modification of cell surface, drug efflux
system and existence of genes associated to certain plasmids (Fange
et al., 2009). This clearly showed that there is a need to continue
research on E. coli strains to able to characterize the multidrug resistant
strains and to understand the actual reasons of the antibiotic resistance phenomena
which is many cases even at modification of cell membrane or secretion of enzymes
and modifications of plasmid associated genes (Harvey and
The present study was undertaken to isolate and characterize the multidrug
resistant strains of E. coli from Egyptian patients by possible biochemical
and molecular methods. The study was also aimed to study the genetic determinants
of the antibiotic resistance ability in E. coli UR4.
MATERIALS AND METHODS
Isolation of bacterial cultures and antibiotic sensitivity test: About
50 clinical samples of urine, blood and stool were taken from patients who did
not respond to treatment by antibiotics Egyptian patients and were transferred
asceptically to the Microbiology Lab. Bacterial cultures were isolated onto
MacConkey agar media (Oxoid) and Hichrome E. coli selective media (HiCrome,TM).
Cultures grown on both media were predicted as Escherichia bacteria and
were consequently, chosen for study. The antibiotic bioassay were studied using
disc diffusion assay for 17 bacterial isolates grown on Hichrome media against
14 types of antibiotics listed in Table 1 (Ehinmidu,
2003). Results were taken according the manufacturers instructions
of antibiotic discs (Oxoid).
Identification of multidrug resistant bacteria: Gram staining, cell
morphology, motility, catalase reaction, oxidase test, production of H2S
and growth of bacteria on both MacConkey agar and Hichrome agar confirmed that
the 17 bacterial isolates belonged to genus Escherichia. The species
was determined by tests listed in Table 2 (Wolfgang
et al., 1998). Identification was carried out according to diagnostic
key of Holt et al. (1994) and Garrity
et al. (2005).
The E. coli UR4 strain which was resistant to all antibiotics used were
subjected to molecular characterization by elucidation of 16 S rRNA cataloging
|| Antibiotic sensitivity test of 17 bacterial isolates from
many patients at two hospitals in Cairo
|R: Resistant bacteria, I: Intermediate, S: Sensitive
|| Biochemical characteristics of the antibiotics resistant
DNA was extracted from UR4 strain according to protocol of Liu
et al. (2002). PCR mediated amplification of the 16 S r RNA gene
and its purification were done using PrepMan Ultra (Applied Biosystem), Microseq
PCR and Miroseq Cycle Sequencing Applied Biosystem. The 16 S rRNA gene was amplified
with primer mixture viz., 5'AGAGTTT GATCMTGGCTCAG '3 forward primer and 3' TACAG
CATTGTTCCATTGGCAT '5 reverse primer.
The PCR mixture consisted of 30 picomoles of each primer, 10 ng of chromosomal
DNA, 200 μM dNTPs and 2.5 Units of Taq polymerase in 50 μL of polymerase
buffer. The PCR was carried out for 30 cycles in 94°C for 1 min., 55°C
for 1 min and 72°C for 2 min. After completion, a fraction of the PCR mixture
was examined using agarose gel electrophoresis (Ausubel et
al., 1999) and the remnant was purified using QIA quick PCR purification
reagents (Qiagen). DNA sequences were obtained using a 3130 X DNA sequencer
(Genetic Analyzer, Applied Biosystems, Hitachi, Japan), BigDye Terminator Cycle
Sequencing (see details below). The PCR product was sequenced using the same
PCR primers. Blast program was used to assess the DNA similarities and multiple
sequence alignment and molecular phylogeny were performed using Bio Edit software
DNA sequencing: Automated DNA sequencing based on enzymatic chain terminator
technique, developed by Sanger et al. (1977)
was done using 3130 X DNA Sequencer (Genetic Analyzer, Applied Biosystems, Hitachi,
Japan). The sequencing reaction was performed with four different fluorescent
labels identifying the ddNTPs, instead of the radioactive labels. These flurophores
were excited with two argon lasers at 488 and 514 nm, respectively when the
respective bands passed the lasers during the electrophoresis. The specific
emissions were detected and the data were collected for analysis (Prober
et al., 1987 and Freeman et al., 1990).
The thermal cycling mixture was as follows: 8 μL of BigDye terminator mix,
6 μL of the sequencing primer (10 pmol) and 6 μL of the PCR product,
then the reaction was run in the thermal cycler. The cyclic reaction composed
of 1 min at 95°C, then 49 cycles of 30 sec at 95°C, 10 sec at 52°C
and 4 min at 60°C. The products were purified using special column according
to the instruction of the manufacturer. The eluates were taken and add high
dye formamide with (1:1)/volume ratio, run at 95°C for 5 min for denaturation,
shock on ice; then the sample become ready for sequencing in 3130 X DNA sequencer
and analysis. The sequence of 16 S gene was sent to glue bank under accession
Selection of E. coli UR4 mutants: Elimination of the antibiotic
resistance ability was carried out according to Enan et
al. (1996). A series of test tubes, each containing 9 mL nutrient broth
with or without 20 mg mL-1 of each of the curing agents or antibiotics
listed in Table 3, were inoculated with log phase cells of
the E. coli UR4 wild strain to give 2x107 CFU mL-1.
Tubes supplemented with curing agents; tubes without curing agents were incubated
at 30 and 42°C, respectively for 48 h. Tubes containing visible bacterial
growth were transferred another time in another tubes like the first rounds
of the experiment. After successive transfer, bacterial suspensions were plated
onto nutrient agar plates and were counted. Number of colonies sensitive to
the cold solution I [50 mM glucose, 25 mM Tris buffer (pH antibiotics listed
in Table 4 were counted after their antibiotic bioassay onto
separate nutrient agar plates (Enan et al., 1996).
Plasmid isolation and agarose gel electrophoresis: Plasmids were extracted
from UR4 wild strain and its mutants according to Sambrook
et al. (1989):
||Multiple colonies were collected using a sterile loop and
added to individual tubes containing 2 mL of nutrient broth, incubated overnight
||One and half mililitter of the culture was poured into microcentrifuge
tube and centrifuged at 12,000 rpm for 30 sec at 4°C
||The supernatant was removed, leaving the bacterial pellet as dry as possible
||The bacterial pellet was resuspnded in 100 μL of ice 8.0), 10 mM
EDTA (pH 8.0)]. Vigorous vortexing was done to ensure that the pellet was
completely dispersed in solution 1
||Two hundred microlitter of freshly prepared solution II [0.2 NaOH in 1%
SDS) were added and the tube was closed tightly, inverted rapidly five times
and stored on ice for 5 min. At this stage a viscous bacterial lysate was
||One hundred and fifty μL of ice cold solution III [3M potassium acetate
in 11.5% glacial acetic acid] were added to neutralize the reaction and
the tubes were closed and vortexed for 10 sec to disperse solution III through
the viscous bacterial lysate then stored on ice for 3-5 min
||The tubes were centrifuged at 12,000 rpm for 5 min at 4°C then the
supernatant was transferred to a fresh tube
||Two volumes of ethanol (double the volume of the supernatant) were added
(to precipitate the double stranded DNA) and mixed by votexing. The mixture
was allowed to stand at room temperature for 2 min
||The tubes were centrifuged at 12,000 rpm for 5 min at 4°C
||The supernatant was removed and the tubes were inverted on a paper towel
to drain away all fluids
||The pellets were rinsed with 1 mL of 70% ethanol and allowed to dry in
the air for 10 min
||The pellets were redissolved in 50 μL of tris EDTA (TE) buffer (10
mM Tris HCl pH 8.0 in 1 mM EDTA), vortexed briefly and stored at 20°C
|| Biochemical characteristics of the wild E. coli UR4
strain and its mutants
Agarose gel electrophoresis was carried out according to Davis
et al. (1986). Twenty five microliter of tracking dye were added
to 100 μL of plasmid DNA preparation. The sample was loaded on 0.7% agarose
gel in Tris-EDTA buffer pH 8.2 and electrophoresis was conducted at 100 volt
for 1-2 h. The gel was stained is EtBr (0.5 g mL-1) for 15 min.,
distained in 1 mM MgSO4 for 30 min, photographed by gel documentation
Isolation, characterization and identification of multidrug resistant bacteria: Urine, blood and stool samples were taken from patients who did not respond to medications and were analysed microbiologically onto both MacConkey agar and HiCromeTM agar media. About 17 culture grown well were chosen and were assayed against 14 types of antibiotics listed in Table 1. All the 17 bacterial cultures tested were showed to be multidrug resistant bacterial cultures.
All the 17 bacterial cultures were rod shaped, motile, Gram negative and catalase
positive cells. Many biochemical tests used for bacterial identification were
studied. Results are given in Table 2. All isolates showed
positive results with regard to indole test, methyl red test, liquefaction of
gelation, utilization of glucose, lactose, maltose, mannitol, L. arabinose,
D-sorbitol and blood hemolysis; but showed negative results with other biochemical
tests listed in Table 2. Following diagnostic key of Holt
et al. (1994) and Garrity et al. (2005),
all the 17 bacterial isolates were identified as belonging to E. coli.
Since, UR4 strain was resistant to all the 14 antibiotics used, it was important to characterize it by 16 S rRNA cataloging analysis for more description of this interesting multidrug resistant bacterium. DNA was isolated from this strain and the 16 S rRNA gene was amplified by PCR technique. Amplified 16 S gene was electrophoresed using agarose gel and the clear band of 16 S gene was taken and sequenced (Fig. 1). The sequence of 16 S gene was submitted to gene bank under accession number JQ388918. It was showed 99.5% similarity to E. coli category.
Selection of antibiotic resistance negative clones: To produce an antibiotic resistance negative clones, the UR4 strain was subjected to 20 mg mL-1 of antibiotics, SDS, sodium azide and ethidium bromide and was grown at elevated temperature (42°C). The results are presented in Table 3. In population treated with antibiotic, no negative clones (sensitive colonies to antibiotic used) were observed. There was no dependence of the number of either antibiotic resistance negative clones or antibiotic resistance positive clones on the applied dose (20 mg mL-1) of the antibiotic used.
However, the occurrence of an antibiotic resistance negative colonies (sensitive colonies to the antibiotic) was found in populations either treated with curing agents or grown at elevated temperature. The mutation percentage was 0.61; 1.24; 0.74 and 14.85% in the experimental antibiotic resistant cells of E. coli UR4 after using the curing agents viz SDS; elevated temperature sodium azide; ethidium bromide, respectively. The produced mutants of the antibiotic resistance negative colones became sensitive to many antibiotics (Fig. 2). The mutants produced by SDS; elevated temperature; sodium azide; ethidium bromide were termed SDS; ET; SAZ; ETH and were used for further experiments.
||Sequence of 16S rDNA gene of the wild strain E. coli
||Antibiotic sensitivity test of the E. coli UR4 mutants
produced after curing with, (a) SDS, (b) Elevated temperature, (c) Sodium
azide and (d) Ethidium bromide
Stability of mutation in the antibiotic resistance negative clones: SDS, ET, SAZ and ETH, were subcultured ten times in nutrient broth. After each subculture, antibiotic sensitively test was carried out. None of them was antibiotic resistant again but all colonies were sensitive to some antibiotic used (Fig. 2).
Comparison of the E. coli UR4 wild strain and its mutants: The antibiotic resistant wild strain E. coli UR4 and its mutants SDS, ET, SAZ and ETH which became sensitive to the antibiotic used were grown in nutrient broth. Growth values were compared. No differences in the rate of growth were observed.
Comparison of biochemical characteristics of the E. coli UR4 wild strain and its mutants: E. coli UR4 wild strain and its mutants SDS, ET, SAZ and ETH which became sensitive clones were subjected to many biochemical tests used for bacterial identification Table 4. Results were compared. There was a quite similar results with regard to biochemical tests listed in Table 4.
Plasmid profile: To study the genetic linkage of E. coli UR4, it mandatory to compare between the plasmid content of the wild strain of E. coli UR4 and its mutants SDS, ET, SAZ and ETH to elucidate whether antibiotic resistance ability is plasmid encoded or is linked to gene located on the chromosome. If the antibiotic resistance ability is plasmid encoded, one plasmid could be deficient in the antibiotic resistance negative variant. However, in case of the antibiotic resistance is chromosomal encoded, the plasmid profile of the wild strain and each mutant should be similar to each other.
Since, the E. coli UR4 wild strain and its mutants SDS, ET SAZ and ETH
which became sensitive to antibiotics used had a quite similar physiological
properties, plasmids were isolated of the wild strain of E. coli UR4
and its mutants SDS, ET, SAZ and ETH. A preparations of Lumda DNA (Omega) were
used as standards. Plasmids and standard DNA were electrophoresed using agrose
gel.; plasmid profile is given in (Fig. 3). The plasmid profile
of the UR4 wild strain is shown in lane 1 and appears to contain five plasmids
with a molecular mass range from 80-1031 bp.
||Agrose gel electrophoresis of plasmids extracted from
E. coli UR4, Lane 1: Wild strain, Lane 2: Mutant strains ET, Lane 3:
SDS, Lane 4: Ethidium bromide, Lane 5: SAZ
The SDS, ET, SAZ and ETH mutants appeared to contain only four plasmids. One
plasmid from each mutant of a molecular mass of about 600 bp was lost or dissociated
in lane 2, 3, 4 and 5. This clearly indicated that this plasmid is the responsible
one on the antibiotic resistance ability in the experimental E. coli
UR4 strain and this antibiotic resistance ability appeared herein as plasmid
E. coli is a part of the normal flora of the human colon but can be
pathogenic both within and outside of the gastrointestinal tract. The difference
of the degree of virulence of different E. coli strains are caused by
individual plasmid associated with each strain. Many strains of E. coli
are pathogenic such as enteropathogenic; enterotoxigenic; enterohemorrhagic;
enteroinvasive; enteroadherant which cause watery diarrhea; blood diarrhea;
colitis; persistant watery diarrhea in children, respectively (Harvey
and Champe, 2012. This clearly showed that there is a need to continue research
on E. coli to control its growth for healthy reasons. Unfortunately,
the existence of the antibiotic resistance ability in E. coli was previously
shown by many authors (Whittam et al., 1993;
Todar, 2007). Resistance of E. coli to antibiotic
was in many reports due to genetic reasons and this clearly showed that there
is a need to study this phenomena to able be find out new control strategies
on the antibiotic resistant strains of E. coli.
In this study, 17 bacterial cultures were isolated on specific Hichrome medium
from Egyptian patient suffering from urinogential infection symptoms even after
antibiotic treatment. It was of interest to study such bacterial cultures. The
17 bacterial cultures appeared to be multidrug resistant bacteria. This supported
latter work in this respect(Ehinmidu, 2003). Since this
phenomena is very necessary clinically, isolates were identified as E. coli
UR4 (Holt et al., 1994; Garrity
et al., 2005). For further characterization of the most antibiotic
resistant bacterium (UR4 strain), the molecular identification by 16 S r RNA
gene sequence analysis was made and UR4 strain appeared to be similar to E.
coli sequence in gene bank (Pot and Janssen, 1993).
Later on, it was of interest to study the genetic determinants of the antibiotic
resistance ability in the UR4. This to concur with previous study in this respect
( Enan and Saad, 2000).
Growing of the experimental UR4 and treatment of its cells with some curing
agents led to mutants lost the antibiotic resistance ability and similar work
was published by Lichstein and Soule (1994). The mutation
was stable character and the SDS, ET, SAZ and ETH mutants continued sensitive
to most antibiotic used and no recovery was observed in any of the above mutants.
This support latter findings in this respect. The wild E. coli strain
contained five plasmids and this clearly indicated the genetic structure of
this strain which expresses on high virulence and high ability of this strain
to resistant all antibiotics used. This is in agreement with Schafer
et al. (1999). The SDS, ET, SAZ and ETH mutants lacked one plasmid
of molecular region of about 600 bp and this clearly indicated the role of this
plasmid in the antibiotic resistance ability of E. coli UR4. Hence the
antibiotic resistance ability in this strain was plasmid encoded.
Further study will be necessary to digest the plasmid containing the antibiotic resistance gene by restriction enzymes to understand its genetic map and to its modifications.
The 17 antibiotic bacterial isolates were isolated, characterized by phenotypic and biochemical criteria. They were identified as belonging to E .coli. One only of them was resistant to 14 antibiotics used; was further characterized by 16Sr RNA sequence analysis. It was identified and designated E .coli UR4. Mutants of such strain were obtained by some curing agents; and the plasmids of UR4 wild strain and its mutants were obtained. One plasmid of about 600 bp was showed to be the committed genetic factor for the antibiotic resistance ability in the E .coli UR4 strain.
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