Journal of Animal Health and Production
January 2016 | Volume 4 | Issue 1 | Page 1
Poultry farming is widely adopted in Pakistan and al-
most every farmstead keeps some poultry mainly for
consumption and cash sales. e science and technolo-
gy have contributed widely for the expansion of poultry
industry and a number of strategies have been adopted
to modulate the quality of poultry products (Abel et al.,
2014). In Pakistan, there are about 25000 poultry farms,
providing employment and income for livelihood of fteen
thousand people. In the country, there are 400 hatcheries,
150 feed mills, 8.5 million broiler breeders, 0.428 million
layer breeders and their feed consumption is 5.51 million
metric tons per year (Anonyms, 2011; FAO, 2011).
E. coli are one of the common microbial ora of gut of
farm animals, poultry and human being. Most of E. coli
isolates are harmless, however, some strains are pathogenic
and may cause serious food poisoning in human beings
(Begum et al., 2014). A recent survey about prevalence of
virulence E. coli based on Congo red binding ability have
indicated more than 90% isolates as pathogenic (Yadav et
al., 2014). In past two decades, severe outbreaks with gas-
trointestinal symptoms have been occurred by food borne
pathogenic E. coli, particularly 0157:H7 (Armstrong et al.,
1996). E. coliand its related species are named as “enteric
bacteria”; because they mostly live in the intestinal tracts
of human and other animal species (Minnock et al., 2000).
About 10 to 15% of intestinal coliforms are opportunistic
and pathogenic serotypes and cause a variety of lesions in
immuno-compromised hosts including poultry (Daini et
al., 2008; Mailk et al., 2013); and may cause omphalitis,
yolk sac infection, cellulitis, colibacillosis and swollen head
syndrome (Gross, 1994).
Table eggs are the primary source of protein in human diet.
ese are used in a number of traditional Pakistani dishes
from decades. However, the recent studies have declared
Abstract | Escherichia coli is one of the common microbial ora of poultry gut. Most of E. coli isolates are nonpath-
ogenic but are considered to be an indicator of fecal contamination in food industry. A study was carried-out on the
prevalence, incidence, isolation and antibiogram of E. coli from table eggs. A total of 100 table eggs were collected from
various locations of district Peshawar, Pakistan and divided into three parts viz., the egg-yolk, egg-white and eggshell.
ese were cultured on dierent media and identied organism was subjected to antibiogram study using the disk
diusion method. e overall prevalence of E. coli was found as 37%. While, incidence was recorded as 15% in egg-
shells, 12% in egg-whites and 10% in egg-yolks. It was concluded that the table eggs were contaminated with E. coli
and higher incidence of E. coli was recorded in eggshells as compared to other components of the eggs. e antibiotics
ciprooxacin and enrooxacin were recorded highly active against E. coli.
Keywords | Escherichia coli, Egg-shell, Egg-yolk, Prevalence, Egg-white, Antibiogram, Peshawar
AurAngzeb KhAn1*, rAhmAtullAh rind1, muhAmmAd ShoAib1, ASghAr Ali KAmboh1, gulfAm
Ali mughAl2, ShAKeel AhmAd lAKho1, KAnwAr KumAr mAlhi1, Ali rAzA nizAmAni3, AdnAn
Isolation, Identication and Antibiogram of Escherichia coli from
Editor | Sanjay Kumar Singh, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly (UP), India.
Received | October 13, 2015; Revised | November 03, 2015; Accepted | November 06, 2015; Published | December 12, 2015
*Correspondence | Muhammad Shoaib, Department of Veterinary Microbiology, Sindh Agriculture University, Tandojam, Pakistan; Email: vet.socialray@gmail.
Citation | Khan A, Rind R, Shoaib M, Kamboh AA, Mughal GA, Lakho SA, Malhi KK, Nizamani AR, Yousaf A (2016). Isolation, identication and antibio-
gram of Escherichia coli from table eggs. J. Anim. Health Prod. 4(1): 1-5.
DOI | http://dx.doi.org/10.14737/journal.jahp/2016/184.108.40.206
ISSN | 2308–2801
Copyright © 2016 Khan et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.
1Department of Veterinary Microbiology; 2Department of Livestock Management; 3Department of Veterinary Pa-
thology; 4Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam, Pakistan.
Journal of Animal Health and Production
January 2016 | Volume 4 | Issue 1 | Page 2
that enteric bacteria like Salmonella, E. coli, Listeria, etc.,
could contaminate these eggs and may cause egg-borne
diseases (Adesiyun et al., 2006; Adesiyun et al., 2007).
Some global epidemics have also been linked with egg
consumption and known to cause egg-borne pathogens
present in poultry eggs and their contents (CDC, 1990;
Rocourt et al., 2003). Food poisoning associated with egg-
borne pathogens may cause severe morbidity or mortali-
ty with diarrhea, vomiting, nausea and abdominal cramps
(Mitchell, 2005). e present investigation was therefore,
designed to study the prevalence and incidence of E. coli in
table eggs sold in retail market of district Peshawar. More-
over, the antibiogram study of isolated E. coli from poultry
eggs was also carried out to investigate the susceptibility
pattern of various antibiotics.
MATERIALS AND METHODS
A total of one hundred poultry table eggs were collected
randomly from dierent markets existed in various local-
ities of district Peshawar, Khyber Pakhtunkhwa, Pakistan.
Eggs were collected from four dierent localities (n= 25
from each locality) i.e., Bacha Khan Chowk, Karkhano
Road, Nahaqi and Palossi Markets of Peshawar. Although,
the eggs were kept at room temperature at sale outlets, so it
was ensured that these should not be older than 24 hours.
Moreover, the eggs with visible fecal shell contamination
were not taken as samples. e collected eggs were trans-
ported to laboratory under cold chain and were kept in
refrigerator at 4°C until they were processed for microbial
For the isolation of E. coli, table eggs were processed ac-
cording to procedure described by Adesiyun et al., (2006).
In brief, using aseptic conditions one sterile swab mois-
tened in normal saline (0.9% NaCl w/v) was applied to the
surface of each egg. It was dipped in 1ml saline in universal
bottle to form a representative egg shell sample. For egg-
yolk and egg-white samples, the eggs were immersed in
75% ethanol for 5 minutes and then pointed end of each
egg was disinfected on Bunsen burner ame for 5-10 sec-
onds. en, a small hole was made on the shell surface and
the egg-yolk and egg-white were emptied separately into
the sterilized polythene bags. e contents were blended
manually. e resultant mixtures and egg shell samples
were used for bacteriological culture as described earlier
(Nazia et al., 2015). e isolated E. coli were then subjected
towards dierent biochemical and sugar fermentation tests
for species conrmation like starch test, lipid hydrolysis
test, casein hydrolysis test, gelatin hydrolysis, carbohydrate
fermentation test, triple sugar iron test, which were based
on their capability to breakdown complex molecules in to
simpler nutritional elements.
In-vItro SuScePtibilitY of E. coli to
e antibiotics (Difco, Michigan, USA) used during the
study were amoxicillin (10ug), colistin (10ug), gentamycin
(10ug), enrooxacin (05ug), kanamycin (10ug), ciproox-
acin (05ug), noroxacin (10ug), tetracycline (30ug) and
doxycycline (30ug). All the E. coli isolates of table eggs
were investigated for their in-vitro susceptibility pattern to
various antimicrobial agents using disk diusion method as
described by Bauer et al. (1966). In brief, the Muller Hin-
ton agar (Difco, Michigan, USA) was prepared, dispensed
in Petri dishes and surface was dried by incubating at 37oC
for 30 minutes. e isolated colonies were selected and sus-
pended evenly in 4ml sterile normal saline solution (0.9%
NaCl w/v; pH: 7.0). A sterile cotton swab was dipped into
the suspension and culture was smeared on the surface of
Muller Hinton agar in such a way that all agar surfaces
would be covered evenly with the bacterial suspension. e
plates were then placed in incubator for 30 minutes to get
dried. e antibiotic discs were placed on the agar surface
with the disc disperser and slightly pressed with sterile
forceps to keep it adhere to the surface. e plates were
then closed, wrapped in polythene bag, inverted in such
a way that medium and discs would be in upward portion
and placed in incubator for 24 hours at 37oC. e zones of
inhibition were observed as a clear area, free from growth
around the discs. Clear zones of inhibition made against
organism by the antibiotics were recorded in mm from the
centre of disc of zone with the observed annotations.
All the experimental results were calculated and presented
in percentage format using the Excel Spreadsheets.
Of the total 100 table eggs examined, the overall prevalence
of E. coli was recorded as 37.00%, while 63.00% eggs were
found free from E. coli contamination (Table 1). Of the 25
eggs examined from Bacha Khan Chowk, the prevalence
of the E. coli species was noted in 40.00% eggs. Similarly,
25 eggs acquired from Karkhano Road market, the prev-
alence was observed as 48.00%. When the same number
eggs were examined from Nahaqi market, the prevalence
of E. coli was recorded as 32.00% in eggs. Whereas 25 eggs
collected from Palossi market showed the prevalence of E.
coli as 28.00% (Table 2).
e results regarding the incidence of E. coli in dierent
components of eggs has been summarized in Table 3. Of
the 100 egg shells examined, the incidence was recorded as
15.00%. Similarly, among 100 egg-whites, the incidence of
E. coli was noted as 12.00%, whereas within 100 egg-yolks
E. coli was detected in 10.00% egg-yolks.
Journal of Animal Health and Production
January 2016 | Volume 4 | Issue 1 | Page 3
Table 1: e overall prevalence of Escherichia coli in table eggs collected from retail markets of Peshawar
Total No. of eggs examined No. of eggs positive % of eggs positive No. of eggs negative % of eggs negative
100 37 37 63 63
Table 2: e number and percentage prevalence of Escherichia coli in table eggs collected from dierent localities of
S. No Name of area Total No. of eggs
Total No. of
% of positive
Total No. of
% of negative
1 Bacha Khan Chowk 25 10 40 15 60
2 Karkhano Road 25 12 48 13 52
3 Nahaqi 25 8 32 17 68
4 Palossi 25 7 28 18 72
Table 3: e number and percentage incidence of Escherichia coli in dierent components of table eggs
Total No. of egg com-
Number of posi-
Percentage of posi-
Number of nega-
Percentage of nega-
Egg-yolk 100 10 10.00 90 90.00
Egg-white 100 12 12.00 88 88.00
Egg-shell 100 15 15.00 85 85.00
Table 4: Antibiogram results of Escherichia coli isolates of
2 mm + Weakly sen-
8 mm +++ Quite sensitive
10 mm +++ Quite sensitive
14 mm ++++ Highly sensi-
2 mm + Weakly sen-
14mm ++++ Highly sensi-
4 mm ++ Moderately
0 mm - Not sensitive
3 mm ++ Moderately
-: Absence of clear zones around disc; +: clear zone up to 2mm; ++:
clear zone with >2-5 mm; +++: clear zone with >5-10 mm; ++++:
clear zone with >10-15mm
During present experiments, nine dierent antibiotics
were tested to demonstrate the in-vitro susceptibility of
E. coli isolates recognized from the table eggs and results
were given in Table 4. e antibiotics ciprooxacin and
enrooxacin were recorded as highly active against E. coli
isolates and inhibited its growth, while antibiotics colistin
and gentamycin were recorded as quite active against E.
coli. Whereas, drugs noroxacin and doxycycline showed
moderate sensitivity against E. coli, as these drugs inhib-
ited the growth of the organisms and showed small zones
of inhibition (>2-5mm) around the discs. Furthermore,
the antibiotics amoxicillin and kanamycin were marked as
weakly active against the organism. However, the antibi-
otic tetracycline failed to inhibit the growth of bacterial
organism on agar plate and was recorded as completely re-
sistant against E. coli.
It has been estimated that many nutrient substances found
in table eggs create an excellent environment for the
growth and development of potential spoilage or infectious
microorganisms. Present study has demonstrated an over-
all 37.00% E. coli contamination in table eggs. is nd-
ing is in agreement with a study conducted by Adesiyun et
al. (2006) in Trinidad. e researchers reported a 71/184
(38.6%) table eggs positive for enteric microbes including
E. coli, Salmonella, etc. Likewise another Polish study re-
ported a 40.30% bacterial contamination in table eggs with
E. coli as most dominant contaminant (Stępień-Pyśniak,
We got 28-48% contamination of bacterial organism in
table eggs collected from dierent localities of Peshawar.
However, another study reported the 36.3 to 69.6% con-
tamination in poultry eggs collected from dierent points
i.e., supermarket, mall and farm (Adesiyun et al., 2006).
ese dierences might be due to dierence in man-
agement, handling and hygienic conditions used at farm
and/or sale outlets. e poultry eggs can get contamina-
Journal of Animal Health and Production
January 2016 | Volume 4 | Issue 1 | Page 4
tion either horizontally (through the shell) or vertically
(trans-ovarial), and could serve a potential source of path-
ogens participating in the etiology of foodborne diseas-
es (Stępień-Pyśniak, 2010). Indar et al. (1998) reported
trans-ovarial transmission of Salmonella spp. in table eggs
collected from commercial poultry farms in Trinidad.
Although, egg-yolk contains maternal immunoglobulin
IgG (also called IgY), but its level could be inuenced by
various factors like, functional quality of immunological
system and/or antibiotics exposure to fowl (Tokarzewski,
2002). Moreover the quantitative contamination of eggs
depends upon bacterial load in the environment where
eggs laid and/or handled (Stępień-Pyśniak, 2010).
e results of the present study indicated the bacterial
contamination level as 15, 12 and 10% on eggshells, egg-
whites and egg-yolk respectively. Adesiyun et al. (2006)
reported in their investigation the contamination level as
19% and 13% in eggshell and egg contents respectively. It
has been suggested that temperature, and/or storage con-
ditions provided to the eggs at retail outlets signicantly
impact the bacterial load of eggs without aecting the bac-
terial prevalence (Suresh et al., 2005). In consistent with
this study, Stępień-Pyśniak (2010) also reported a high
contamination level of eggshells as compared to other in-
ternal contents. is is probably due to exposure of egg-
shell with the environment.
e results about in-vitro susceptibility of E. coli isolat-
ed from table eggs of poultry birds recorded during pres-
ent investigation were in line to the ndings reported by
previous studies. Like, Akond et al. (2009) isolated and
identied E. coli from poultry sources of dierent poul-
try markets and sensitivity to antimicrobials was recorded
as 86, 80, 60, 36, 30 and 26% to noroxacin, gentamicin
and chloramphenicol, neomycin, tetracycline, streptomy-
cin and ampicillin, respectively. Raji et al. (2007) observed
ciprooxacin as highly active (85-100%) antibiotic against
E. coli isolates. Adesiyun et al. (2007) studied the resist-
ance of bacterial species to seven antimicrobial agents us-
ing the Disc Diusion Method. An overall, 131 bacterial
isolates of E. coli and Enterobacteriaceae were tested, and
125 (95.4%) exhibited resistance to one or more antimi-
crobial agents. e high resistance was recorded against
streptomycin (90.1%), tetracycline (51.9%) and kanamy-
In present investigation we have found 3/9 (33.33%) an-
timicrobial agents as resistant or weakly sensitive to E. coli
isolated from table eggs. is nding is in agreement with
a study conducted by Musgrove et al. (2006). e study in-
dicated that most (73.2%) of E. coli isolated from eggshells
were susceptible to all antimicrobial agents. Moreover, the
E. coli isolates showed 29.9, 6.2 and 3.1% resistance to tet-
racycline, streptomycin and gentamicin respectively. Simi-
lar results were also reported by Ansari et al. (2014).
It could be concluded from present investigation that table
eggs sold in retail market of district Peshawar contained
E. coli, hence may pose a health hazard to human beings
if consumed improperly cooked or raw eggs. Eggshells
contained more bacterial contaminants as compared to
egg contents. Antimicrobial agent tetracycline was found
completely resistant to E. coli isolates, whereas, amoxicillin
and kanamycin were observed as weakly sensitive. ere is
a need to educate the people to adopt signicant hygienic
measures in handling of table eggs and should not be con-
sumed inadequately cooked eggs or egg products.
e author is thankful to the honourable Supervisor, Dr.
Rahmatullah Rind, Professor, Department of Veterinary
Microbiology, Faculty of Animal Husbandry and Veter-
inary Sciences, Sindh Agriculture University, Tandojam,
for his courteous professional pieces of advice, untiring
guidance, full support, motivation, fruitful suggestions and
encouragement during the whole period of study and also
thankful to the respectable and sincere teacher Dr. Asghar
Ali Kamboh, Assistant professor, Department of Veteri-
nary Microbiology SAU Tandojam for his technical facili-
tation and all others for their co-operation during research
CONFLICT OF INTEREST
ere is no conict of interest.
Mr. Aurangzeb was the main researcher, Dr. Rahmatullah
Rind was his supervisor, Dr. Asghar Ali Kamboh revised
the article, Muhammad Shoaib did all the correspondence,
Gulfam Ali Mughal, Shakeel Ahmad Lakho, Kanwar Ku-
mar Malhi, Ali Raza Nizamani and Adnan Yousaf con-
tributions in statistics, and other activities related to the
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