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JOURNAL OF PURE AND APPLIED MICROBIOLOGY, Dec. 2017. Vol. 11(4), p. 1749-1755
Antibiotics and Phage Sensitivity as Interventions for
Controlling Escherichia coli Isolated from Clinical Specimens
Mamdouh M. Esmat1, Ahmed G. Abdelhamid2, Sabah A. Abo-ELmaaty2,
Mohamed A. Nasr-Eldin2, Mervat G. Hassan2, Abeer A. Khattab2 and
Ahmed Esmael2*
1Medical Microbiology and Immunology Department, Faculty of Medicine,
Sohag University, Sohag, Egypt.
2Botany Department, Faculty of Science, Benha University,
Qalubiya Governorate, 13511, Egypt.
doi:
(Received: 03 October 2017; accepted: 09 November 2017)
Escherichia coli is considered one of the most frequent causative agents of common
bacterial infections worldwide. In addition, effective treatment and prevention measures are
still in demand due to the rise of antibiotic resistance and the emergence of new virulent strains.
In this work, we evaluated antibiotics and bacteriophages as interventions for controlling
pathogenic E. coli. A total of 15 E. coli isolates were included in this study. The automated
identification system, namely Vitek 2, has been utilized for the identification. Antibiotics
susceptibility profiles of all isolates were confirmed by disc diffusion method. All strains
exhibited resistance at least to one antibiotic (ampicillin) while 13 strains were resistant to
Ampicillin/Sulbactam, Cefazolin, and Ceftriaxone. Except for two strains, no resistance to
Amikacin was observed. On the other hand, bacteriophages designated øEU-3 and øEU-4 were
isolated by single plaque isolation and investigated as antimicrobial agents against pathogenic E.
coli. Phages morphology, determined by transmission electron microscopy, revealed a structure
comprised of a head diameter (71.42 nm) and a tail length (214.28 nm). These features placed
the øEU-3 phage in the family Siphoviridae while øEU-4 belonged to family Myoviridae with
a head diameter (66.6 nm) and a contractile tail length (108.3nm). Phages susceptibilities were
determined by spot test to fifteen E. coli isolates. Coliphage øEU-3 and øEU-4 had narrow
host range. This work described the efficacy of antibiotics and bacteriophages as intervention
strategies to control pathogenic E. coli and paved the way for depth studies to broaden the
antimicrobial spectrum of øEU-3 and øEU-4 phages.
Keywords: Escherichia coli, Antibiotic resistance, Coliphage, Siphoviridae, Myoviridae.
Escherichia coli is a gram-negative
bacterium that is considered a part of the normal
flora inhabiting the intestine and is thought to be
harmless. However, pathogenicity mechanisms
have been exhibited by many strains making
them able to cause diseases in animals and
humans. These diseases can be categorized into
extra-intestinal (urinary tract infections (UTI),
meningitis, septicemia, and pneumonia) and
intestinal (diarrhea)1. Six pathotypes of E. coli
causing intestinal diseases are known based on
the pathogenicity profiles (clinical disease and
virulence factors) and include enterohaemorrhagic
E. coli , enteropathogenic E. coli , enteroinvasive
E. coli, enterotoxigenic E. coli, enteroaggregative
E. coli, diffusely adherent E. coli and the recently
emerged; adherent invasive E. coli2–4. The spread
of prolonged-spectrum beta-lactamase producing
E. coli capable of resisting several antibiotics and
even the emergence of resistance to drugs of last
J PURE APPL MICROBIO, 11(4), DECEMBER 2017.
1750
resort such as colistin has become a major health
threat worldwide and motivated the challenge for
antimicrobial alternatives such as bacteriophages5–7.
Bacteriophages are bacteria-specific
viruses8. Félix d’Hérelle, a co-discoverer of
bacteriophages, was the first to propose “phage
therapy”9, 10 in the early twentieth century, however,
the advent of antibiotics overtook the interest in
phage therapy in that time. Nevertheless, phage
therapy is now back in the headings11 because
of the emergence of new infectious antibiotic-
resistance strains. E. coli phages, termed T-even,
T-odd and Lambda phages, are a group of ds-
DNA bacteriophages that have a head capsid and
tail. Phages of E. coli were firstly isolated from a
raw sewerage by T.L. Rakietenin the late 1930s,
and currently, they are the most studied among
bacteriophages. They are commonly isolated from
fecal samples, sewage, polluted rivers and hospital
wastewater. Life cycles of E. coli phages could be
lysogenic or lytic; however, for phage therapy, only
lytic phages are used12. Currently, phage therapy is
being used in Russia, Georgia, Poland. and U.S.A
The current manuscript was designed to
isolate and identify Gram-negative bacilli from
Sohag University Hospital, Sohag Governorate,
Upper Egypt, and to evaluate their antibiotics
sensitivity patterns, also we tried to isolate host-
specific bacteriophages from sewage water to
evaluate the efficacy of phage therapy against the
isolated E. coli.
MATERIAL AND METHODS
Samples collection and bacterial identication
The present study was done in Sohag
University Hospital; a teaching hospital in Upper
Egypt, with more than 1000 bed capacity, and
Faculty of Science, Benha University, Qalubiya
governorate. Clinical specimens from the intensive
care unit (ICU) were collected. Respiratory (R)
secretions and tracheal aspirates (from intubated
patients) were collected from patients with
respiratory symptoms, urine (U) samples were
collected aseptically in a sterile syringe from the
demarcated site of catheterized patients, and swabs
from surgical site infections (SSI) of patients were
collected from deep incisions after skin antisepsis.
The clinical specimens were collected under
aseptic conditions and transferred immediately
for culture and sensitivity during the period from
March 2016 to February 2017. All Samples were
collected aseptically after 48 hours admission in
ICU; transferred immediately to be inoculated on
routine culture media and incubated aerobically at
37°C for 24 hours. Identification and antimicrobial
sensitivity of the isolated gram-negative bacilli
were done by Vitek 2 system.
Antibiotic susceptibility testing
Gram-nega tive iden tifi cati on and
antimicrobial susceptibility testing cards were
used to determine the susceptibility of the isolates
to different antimicrobial agents. The isolates were
either susceptible, intermediate susceptible or
resistant to the antimicrobials. Susceptibility testing
was confirmed using the disc diffusion (modified
Kirby Bauer) method for the following antibiotics
(Oxoid, UK); Ampicillin (AMP 10 µg), Ampicillin/
Sulbactam (SAM 20 µg), Amikacin (AK 30 µg),
Aztreonam (ATM 30 µg), Cefazolin (CZ 30 µg ,
Cefepime (FEP 30 µg), Ceftriaxone (CRO 30 µg),
Ciprofloxacin (CIP 5 µg), Ertapenem (ETP 10 µg),
Meropenem (MEM 10 µg ), Gentamicin (CN 120
µg), Tobramycin (TM 10 µg), Moxifloxacin (MXF
5 µg), Nitrofurantoin (F 300 µg), Trimethoprim/
Sulphamethoxazole (SXT 25 µg), Tigecycline
(TGC 15 µg), and Imipenem (IPM 10 µg). The
results were inferred according to Clinical and
Laboratory Standards Institute (CLSI) guidelines
(CLSI, 2013).
Demonstration of Sewage E. coli phages
Samples of sewage water were collected
in sterile dark containers from the sewage water
treatment plant at Benha, Qalubiya Governorate,
Egypt. The sample was first filtered through coarse
filter paper to remove the debris, then the filtrate
was serially diluted using 0.85% sterile saline,
subsequently, the samples were filtered using 0.45
µm filters and stored at 4°C.
Isolation and propagation of E. coli phages
Fifty milliliters of filtered wastewater
samples mixed with 50 ml of Luria Bertani (LB)
broth inoculating with the log phase cells of E.
coli. The culture media were incubated at 37°C
for 24 h with speed of 120 rpm for bacteriophage
enrichment. The media were centrifuged and
filtered through a 0.45 µm membrane filter. The
presence of lytic phage in the filtrate was examined
by using the overlayer method as described later.
One hundred µl of the filtrate was mixed
J PURE APPL MICROBIO, 11(4), DECEMBER 2017.
1751
Fig. 1. Electron Micrographs of the isolate phages after
negative staining of viral particles. (a) øEU-3 phage.
(b) øEU-4 phage. The size bar corresponds to 100 nm
with 300 ìl of log phase culture of E. coli isolates
and incubated at 37°C for 30 min. The mixture
was added into a 3.5 ml of molten LB soft top agar
(0.75% agar) which was already cooled down to
50°C, mixed gently and poured onto LB agar plate.
The plates were left to stand at room temperature
for 30 min to allow the top agar to solidify.
The presence of lytic phages was distinguished
according to the shape and size of plaques formed
after incubation of the plates at 37°C for 24hr.
Purification and propagation of E. coli phages were
done by single plaque isolates according to (13).
Phage preparations
Stock lysates for the E. coli phages
candidates were prepared by incubation of purified
phage with the host in LB broth supplemented with
CaCl2 (2 mM) for 24-48 h at 37°C with shaking,
the cultures were centrifuged (6000 rpm, 10 min)
then filter sterilized and the resulting crude lysate
was stored at 4°C.
Phage titering
Serial dilutions of the phages stock lysates
were prepared and aliquots of these dilutions were
incubated with the host at room temperature for
30 min. The incubated aliquots were added to
molten LB soft agar and overlaid on an LB agar
plate. Plates were incubated at 37°C for 24 h and
the resulting plaques forming units (PFUs) were
quantified to determine phage titer.
Spot test
The spot assay was used to assess the
prevalence and the bactericide ability of the
sewage water E. coli phages against all the 15
isolated E. coli strains and was repeated three
times for each bacterium. Five microliters of 45
µm filter-sterilized purified phages were spotted
onto the surface of the plates with the different
E. coli strains. The plates were left to dry and
were inspected for lysis zones after an overnight
incubation at 37°C. A positive spot test appeared
as complete obliteration of the entire drop area,
whereas a negative spot test resulted in the bacterial
lawn growing normally in the region of the spot.
positive samples in the spot tests were confirmed
by plaque assay14.
Transmission electron microscopy (TEM)
Ten microliters of the purified phage
lysate with a high titer were spotted onto carbon-
coated grids and stained with 1% uranyl acetate.
Phages particles were observed under a JOEL-JM-
100-C Transmission Electron Microscope (TEM)
(Japan Electron Optics Laboratory Co., Ltd.) in the
Regional Center of Mycology and Biotechnology
at Al-Azhar University, Egypt.
RESULTS
E. coli isolation and antibiotic susceptibility
proling
In this work, fifteen E. coli were isolated
from clinical samples. They were designated as E.
coli U-1 to E. coli U- 4, E. coli R-1, E. coli SI-1
Table 1. In vitro susceptibility of different E. coli
strains against sewage phages using spot assay
Sample Isolate øEU-3 øEU-4 Two phages
source cocktail
U E. coli U-1 - - -
U E. coli U-2 - - -
U E. coli U-3 + + +
U E. coli U-4 + + +
R E. coli R-1 - - -
SSI E. coli SI-1 - - -
SSI E. coli SI-2 - - -
SSI E. coli SI-3 - - -
SSI E. coli SI-4 - - -
SSI E. coli SI-5 - - -
SSI E. coli SI-6 - - -
SSI E. coli SI-7 - - -
SSI E. coli SI-8 - - -
SSI E. coli SI-9 - - -
SSI E. coli SI-10 - - -
- denotes for resistant, +denotes for susceptible
J PURE APPL MICROBIO, 11(4), DECEMBER 2017.
1752
Table 2. Antibiotic resistance patterns of clinical E. coli isolates
Sources of Isolate AMP SAM CZ CRO FEP ATM ETP IPM MEM AK CN TM CIP MXF TGC F SXT
specimens
U E. coli U-1 R* R R R R R S** S S S S S S S S S S
U E. coli U-2 R R R R I† I S S S S R R R R S S R
U E. coli U-3 R NA NA I NA NA NA NA NA R NA NA R NA NA NA NA
U E. coli U-4 R NA NA R NA NA NA NA NA R NA NA R NA NA NA NA
R E. coli R-1 R R R R R R S S S S S S R R S I R
SSI E. coli SI-1 R R R R R R R R R S S R R R S R S
SSI E. coli SI-2 R R R R R NA†† NA NA S S R R R NA NA S R
SSI E. coli SI-3 R R R R S NA NA NA S S R R R NA NA R R
SSI E. coli SI-4 R R R R R R S S S S S R R R S S S
SSI E. coli SI-5 R R R R R NA NA NA S S S S S NA NA S R
SSI E. coli SI-6 R R R R R NA NA NA S S R R R NA NA NA R
SSI E. coli SI-7 R R R R S I S S S S S S S S S S R
SSI E. coli SI-8 R R R R R NA NA NA R S S R R NA NA S S
SSI E. coli SI-9 R R R R R NA NA NA S S R R R NA NA NA R
SSI E. coli SI-10 R R R R R NA NA NA S S R R R NA NA S R
*denotes for Resistant, **denotes for Susceptible
† Intermediate †† Not tested
Table 3. Dimensions and titration of the isolated phages
Phage Prospected Family Head diameter (nm) Tail length (nm) Phage titer
(PFU/ml)
øEU-3 phage Siphoviridae 71.42 214.28 2 x 107
øEU-4 phage Myoviridae 66.6 108.3 2 x 105
J PURE APPL MICROBIO, 11(4), DECEMBER 2017.
1753
to E. coli SI-10 as they were isolated from the
urine samples, respiratory infections and surgical
site infections, respectively. All strains were
resistant to at least one antibiotic (ampicillin).
Thirteen strains were also resistant to Ampicillin/
Sulbactam, Cefazolin, and Ceftriaxone. Except
for two strains, no resistance to Amikacin was
observed. In addition, all strains were sensitive to
Meropenem except for two strains. Nine strains
were resistant to Aztreonam and six strains
resist to gentamycin. One strain was resistant to
Ertapenem and Imipenem while nine and twelve
strains resisted to tobramycin and Ciprofloxacin,
respectively. Furthermore, nine strains were
resistant to Trimethoprim/ Sulphamethoxazole and
out of six strains investigated for Tigecycline, no
resistance was established. The antibiotic resistance
patterns of all strains were summarized in Table 1.
Bacteriophage susceptibility
To investigate the prevalence of
bacteriophages in municipal sewage water against
the previously isolated bacteria, a spot technique
was performed, and the results were summarized in
Table 2. Out of the fifteen isolated E. coli strains,
only two were shown to be sensitive to sewage
bacteriophages, E. coli U-3 and E. coli U-4, as
judged by spot test and later by plaque assay.
The spot technique served as both an indicative
experiment of bacteriophages presence and gave
an idea of the host range of the resulted phages
(data not shown), single isolated phages, as well
as mixed cocktails of the two phages, were used
in the preceding experiments. The accumulated
data revealed a narrow-host-range of the isolated
phages against the fifteen isolates. Single plaques
were picked from the plates with visible clear
plaques, from E. coli U-3 and E. coli U-4 lawns,
the plaques were then propagated in liquid
cultures. The previous phages, named øEU-3 and
øEU-4 phages, were selected for imaging with
transmission electron microscope.
Morphology and diameters of øEU-3
and øEU-4 phages are shown in Fig.1 and Table3.
Electron micrographs of øEU-3 phage showed an
icosahedral head with a diameter of ~71.42 nm and
a 214.28 nm long non-contractile tail (a character
of Siphoviridae phages). Micrographs of øEU-4
phage showed that it has an icosahedral head
with a diameter of ~66.6 nm and a 108.3 nm long
contractile tail (a character of Myovirdaephages).
DISCUSSION
Over the last decades, the prevalence of
multidrug-resistant bacteria, particularly E. coli
has gained a great interest due to their increasing
rates on a daily basis15. The occurrence of extended-
spectrum Beta-lactamase producing E. coli could
be nearly 63% in many countries while the highest
rates belong to E. coli from ICU patients16, 17.
Our work indicated highly drug-resistant E. coli
could be present in the ICU. Many E. coli isolates
showed resistance to at least five antibiotics. In
previous studies, high resistance to antibiotics
has been described for E. coli isolated from UTI
samples18. Moreover, high frequency of E. coli
resistance to multiple antibiot ics such as penicillin,
co-trimoxazole, and nitrofurantoin has been
documented19. In another study, 170 (43%) of E. coli
isolates were multidrug resistant and showed high
resistance to Ampicillin (82.53%), Amoxycillin-
clavulanic acid (71.90%), Ceftriaxone (66.58%),
Ciprofloxacin (65.82%)20. It was indicated that of
total 328 strains of E. coli, 199 strains exhibited
higher resistance rate to ampicillin, tetracycline,
trimethoprim/sulfamethoxazole and cefazolin, and
34.6% of these E. coli strains exhibited resistance
to at least four antibiotics. Three strains were
resistant to seven antibiotics in the same study.
The treatment of multidrug-resistant pathogens
continues to be a challenging problem. Sustainable
and effective alternative approaches have become
a necessity. One of the most popular approaches
is employing bacteriophages used as anti-infective
agents to circumvent antibiotic resistance.
Phage therapy has various merits over
antibiotics: (1) bacteriolysis mechanisms by phages
differ from antibiotics mechanisms (21) so, it is
very effective against antibiotic-resistant bacteria;
(2) bacteriophages are very specific so, they will
be harmless to other beneficial bacteria in human
guts; (3) Phages undergo mutations so, they can
respond quickly to phage-resistant mutants; (4)
ease of isolation; (5) no side effects are known so
far; (6) high therapeutic index, smaller effective
dose, and a single shot is required; (7) production
of phages are very quick and cheap as compared
to development of a new antibiotic22, 23. The major
obstacles against phage therapy are legislation
which might take years to be approved. Currently,
Russia, Poland, Georgia, China and U.S.A are
J PURE APPL MICROBIO, 11(4), DECEMBER 2017.
1754
using bacteriophages to treat systemic and enteric
diseases that do not respond to conventional
antibiotics. Bacteriophages are ubiquitous in
different watersheds throughout the world and
proposed to be most copious biological objects in
the biosphere24, 25. Bacteriophages existed since the
bacterial cells originated, wherever the bacteria
are found, bacteriophages were found associated.
In current study, we succeeded to isolate lytic
bacteriophages, from sewage water, against two of
the preceding E. coli isolates. Likewise our study,
many researchers have isolated phages against E.
coli from sewage26. The morphology of isolated
E. coli phages, designated bacteriophages øEU-3
and øEU-4, showed that they belong to the family
Siphoviridae and Myoviridae, respectively. Similar
study reported isolation of E. coli siphophages
from stool of pediatric patients with a complaint
of diarrhea27 and others indicated in healthy
subjects, as lambda-like Siphoviridae phages were
mainly isolated from stool samples of healthy
persons28, 29 while stools of diarrhea patients gave
predominantly T4-like Myoviridae. But when using
different indicator cells, different phages were
also isolated from the same stool samples27. Our
results showed that the two isolated phages (øEU-
3 and øEU-4) were investigated for their potential
use as antimicrobial agents against the isolated
multidrug resistant clinical E. coli, whereas, E.
coli U-3, E. coli U-4 isolates were susceptible to
phages cocktail infection, but the rest of studied
isolates revealed different patterns of resistance
to antibiotics as well as phage cocktail which
displayed a narrow host range.
CONCLUSION
This study provided evidence that E. coli
isolated from clinical specimens although being
sensitive to certain antibiotics, they exhibited
resistance to a wide range of other drugs. In
addition, the use of lytic bacteriophage cocktail
as antimicrobial agent against the isolated E. coli
strains revealed the efficacy of the two isolated
phages to control two out of fifteen strains used in
this study and paved the way for future studies to
focus on expanding their antimicrobial spectra by
combining them with other antimicrobials and/or
newly isolated phages.
ACKNOWLEDGEMENT
Funding for this study was partially
supported by a grant from the Scientific Research
Fund (SRF) from Benha University (A.G.A), other
funds were from the Egyptian Ministry of Higher
Education and Scientific Research.
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