Journal of Rural Development and Agriculture (2017) 2(1): 66-71
66
Efficacy of quinolones and cephalosporins against antibiogram
of Escherichia coli isolated from chickens
Ranjhan Ali Lakho
1
, Shahid Hussain Abro
1
, Muhammad Tarique Tunio
2*
, Mohsina Zubair
3
, Rani Abro
4,
Rahmatullah Rind
1
, Riaz Ahmed Leghari
1
, Kanwar Kumar Malhi
1
, Muhammad Rafique Rind
5
, and
Asghar Ali Kamboh
1
Key Message This study evaluates the efficacy of quinolones and cephalosporins against antibiogram of E.
coli isolated from chicken flocks and it reveals that these antibiotics were found to be effective against E. coli.
ABSTRACT Colibacillosis is an acute septicemia disease caused by E. coli producing considerable morbidity
and mortality especially in poultry. Quinolones and cephalosporins have been used in treatment of various
infections. Therefore, this study was designed to evaluate the efficacy of quinolones and cephalosporins
against the antibiogram of E. coli isolated from chicken flocks. The 100 blood samples from liver and intestine
were collected from different poultry vendors surrounding of Tandojam and Hyderabad, Sindh, Pakistan. The
isolated organism was cultured on nutrient and blood agar media. The cultural, morphological and
biochemical characteristics were observed for the confirmation of the isolated organism. The minimum
inhibitory concentration (MIC) of different antibiotics against E. coli was performed by serially diluting
antibiotics ciprofloxacin, metronidazole, cefipime as 0.4 µg/ml, 0.8 µg/ml, 1.6 µg/ml, 3.2 µg/ml and 6.4 µg/ml,
12.5 µg/ml, 25 µg/ml and 50 µg/ml, respectively. The mean zones of inhibition of ciprofloxacin, ofloxacin,
enrofloxacin, norfloxacin, cefepime, ceftazidimine and cefoxitin against the antibiogram of E. coli were
recorded as 14, 11, 12, 11, 11, 11, and 11 mm, respectively. MIC results indicated that ciprofloxacin was found
to be more effective to inhibit the growth of E. coli. It was found that antibiotics of quinolones group namely
ciprofloxacin, enroflaxcin and ofloxacin as well as cephalosporin group namely cefipime, ceftazidime and
cefoxitin were found to be effective to isolate E. coli.
Keywords: Antibiogram, Cephalosporins, Chicken, E. coli, Quinolones
1
Department of Veterinary Microbiology, Faculty of Animal Husbandry and Veterinary Sciences, Sindh
Agriculture University, Tandojam 70060, Pakistan
2
Department of Agricultural Sciences, Faculty of Sciences, Allama Iqbal Open University, Islamabad
3
Department of Chemistry, Preston University, Islamabad
4
Department of Animal Nutrition, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture
University, Tandojam 70060, Pakistan
5
Department of Biotechnology, Sindh Agriculture University, Tandojam 70060, Pakistan
*Corresponding author: Muhammad Tarique Tunio (tarique_tunio@hotmail.com)
To cite this article as: Lakho, R. A., Abro, S. H., Tunio, M. T., Zubair, M., Abro, R., Rind, R., Leghari, R. A., Malhi,
K. K., Rind, M. R., & Kamboh, A. A. (2017). Efficacy of quinolones and cephalosporins against antibiogram of
Escherichia coli isolated from chickens. Journal of Rural Development and Agriculture, 2(1), 66-71.
INTRODUCTION
Pakistan is basically an agricultural country and livestock is considered to be an important sub-sector which
contributes about 52.2% to the value addition in the agriculture sector and 11% in the total GDP (Khan et al.,
2008). In Pakistan, poultry industry is developing during the last couple of decades and contributing in the
increase of GDP. This sector not only produces meat and eggs but also provides large scale employment
opportunities and source of income for the people (Government of Pakistan [GOP], 2014); Anjum et al., 2016).
Microbial agents especially bacterial pathogens produce considerable health and production losses in poultry.
ORIGINAL PAPER
Journal of Rural Development and Agriculture (2017) 2(1): 66-71
67
In Pakistan, colibacillosis is one of devastating diseases that causes heavy economic losses in poultry
industry. This disease produces acute septicemia disease caused by E. coli with heavy motility particularly in
broilers (Shah et al., 2004). E. coli were discovered by German Pediatrician and bacteriologist Theodor
Escherichia in 1885. E. coli is widely distributed in nature and commonly found in normal intestinal tracts of
man, animals and birds. It is a gram negative and rod shape bacterium, there are approximately 100
serotypes that have been recognized but E. coli strains 01, 02 and 078 are the most pathogenic causing severe
infections in poultry and survive for longer time in poultry house. Usually, the organisms are present in the
soil, water, dust, air, feed, litter, feathers and in open surfaces of the poultry farm (Holt et al., 1994). E. coli are
not only harmful for poultry but also hazardous for other animal species as well.
E. coli can cause gastroenteritis, urinary tract infection, calf dysentery or “white scores” neonatal
meningitis, mastitis, cystitis, metritis in cattle, sleeping foal disease in horse. It can cause peritonitis,
pneumonia and in some cases it causes septicemia resulting animal death within 48 hours. It also causes
enterotoxaemia in sheep and goats (Panduranga, 1996). In poultry, E. coli infected birds clinically show
dullness, depression, elevated temperature, inappetence, diarrhea, hemorrhages and lesions on heart,
intestines, proventiculous and gizzard (Cardona & Msoffe, 2009). There is growing concern to transmission
and association of infections in humans. Recently, cephalosporin-resistant E. coli strains isolated from poultry
exhibited phylogenetic relationship with the organism from human workers (Angela et al., 2016). The
intestinal inhabitant commensally bacterial species poses a certain resistance against invasion and
colonization of microbial agents (Pamer, 2016). The irrational use of antibiotics in veterinary practice is a
great concern for treatment of bacterial infections. Also several microorganisms have acquired resistance to
drugs, and decreases therapeutic options in clinical settings (Buffie et al., 2012).
Cephalosporins (β-lactum antibiotics; cefepime, cefotaxime, ceftriaxone and cefuroxime) are derived from
fungus Acremonium. These antibiotics are commonly used in treatment of infection by gram negative
bacteria i.e enterococci and Streptococci pneumonia (Palleres et al., 1993; Dahms et al., 1998). Cephalosporins
induce their biological functions by interaction with bacterial enzymes and β-lactum (Flynn & Edwin, 2013).
Quinolones (ciprofloxacin, ofloxacin, rifampicin, lincomycin, bacitracin, enrofoxacin, nalidixic acid and
norfloxacin) are broad-spectrum antibiotics commonly used in veterinary medicine. It has been reported that
these antibiotics lead to alteration, reduce accumulation and DN grease protection (Oliphant et al., 2002). In
E. coli, quinolones target topoismerase IV for its antimicriobial action (Khodursky et al., 1995). Keeping in
view the good therapeutic results of cephalosporins and quinolones against the gram negative bacteria, this
study was designed to evaluate antimicrobial resistance/sensitivity of E. coli to quinolones and
cephalosporins groups of antibiotics commercially available in market.
MATERIALS AND METHODS
Collection of samples, isolation and identification of the organism
The 100 blood samples of liver and intestine were collected from different poultry vendors in surrounding of
Tandojam and Hyderabad, Sindh, Pakistan. The reference strain of E. coli was obtained from Department of
Microbiology, University of Karachi. The samples were analyzed at Department of Veterinary Microbiology,
Sindh Agriculture University Tandojam, Central Veterinary Diagnostic Laboratory, Tandojam, and Vaccine
Production Unit, Tandojam, Hyderabad, Pakistan. The colonies of E. coli were used to obtain pure culture for
the biochemical properties and sugar utilization efficiencies. The samples were cultured, isolated and
identified using the methods described (Abro et al., 2016a, 2016b). The reference strain of E. coli was
processed for confirming the similar cultural and biochemical characteristics.
Susceptibility test
The Muller Hinton agar (Difco) was prepared according to manufacturer’s instructions and incubated at 37 °C
for 15 min. Bulks of pure culture colonies were suspended in normal saline solution in order to match barium
chloride standard for antibiotic sensitivity. Sterile swab was soaked in the suspended solution and culture
was smeared on the medium. The colonies suspended swab was evenly applied on the surface of the medium
and plates were incubated at 37 °C for 15 min. The antibiotics comprising of quinolones group; ciprofloxacin,
ofloxacin, rifampicin, lincomycin, bacitracin, enrofoxacin, nalidixic acid, norfloxacin and Cephalosporins
Journal of Rural Development and Agriculture (2017) 2(1): 66-71
68
group; cephradin, cefuroxime sodium, cefepime, cephalexin, ceftazidime and cefoxitin, cefotaxime, ceftriaxone
and cefuroxime were used in this investigation. The antibiotic discs were applied on medium surface using
disc dispenser and gently pressed with sterile forceps in order to complete contact with the surface of Muller
Hinton agar. The plates were incubated overnight at 37
o
C. The zone of inhibition was measured as a clear
zone (free from growth around the disc = -) and a clear zone of inhibition formed against E. coli. The zone of
inhibition produced by the drugs was measured from the center of disc to zone in millimeters.
Minimum inhibitory concentration (MIC)
This method is used to determine the minimum inhibitory concentration (MIC) of selected antibiotics. Muller
Hilton broth was prepared in ten flasks, marked as 0.4 ug/ml. 0.8 µg/ml, 1.6 µg/ml, 3.2 µg/ml, 6.4 µg/ml, 12.5
µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml and control (c). The medium was then incubated overnight to check
sterility. Selected drugs (ciprofloxacin, metronidazole and cefapime) were added in above mentioned nine
flasks according to the dilution mark on them and tenth was kept as a control. Forty test tubes measuring 13
× 100 mm plugged with cotton were sterilized in hot air oven at 170
0
C for 2 h. The test tubes were grouped
into four. Test tubes were then filled with medium containing antibiotic at the rate of 5 ml per tube. A single
colony from E. coli incubated in the Miller Hilton broth; following the next day the organism was inoculated
into the test tubes of each antibiotics group comprising of quinolones and cephalosporins. The test tubes
were then incubated over-night and growth was observed. The minimum inhibitory concentration (MIC) of
different antibiotics against E. coli was performed by serially diluting antibiotics as 0.4 µg/ml, 0.8 µg/ml, 1.6
µg/ml, 3.2 µg/ml and 6.4 µg/ml, 12.5 µg/ml, 25 µg/ml and 50 µg/ml, respectively.
RESULTS AND DISCUSSION
The data regarding mean size of inhibition of various antibiotics against E. coli is presented in Table 1. Higher
zones of inhibition were formed by ciprofloxacin, ofloxacin, enrofloxacin, norfloxacin, cefepime, ceftazidimine
and cefoxitin and their mean zones of inhibition was 14.0, 11.0, 12.0, 11.0, 11.0, 11.0 and 11.0 mm,
respectively. Previously, it has been reported that organism is highly sensitive to these antibiotics (Rozina et
al., 2004). However, earlier research observed that increase in resistance of E.coli to different antibiotics;
oxytetracycline (97-100%), tetracycline (95-100%), neomycin (62-71%), trimethoprim (95-98%) and
amoxicillin (50-65%). The exposure of E. coli was detected in chicken flocks of various poultry farms in
Pakistan. The sensitivity of bacitracin against the isolated E. coli was observed resistant therapeutic in the
control of E. coli infection. The findings of Ogunbanwo and Onilude (2004) are contradicted with the findings
of our study. It was revealed in this study that E. coli showed complete resistance against bacitracin. While
other antibiotics showed smaller zone of inhibition and were recorded as moderate or less effective against E.
coli. However, it was observed that E. coli showed complete resistance against the lincomycin and bacitracin
(Table 1). The efficiencies of enrofloxacin (73.96%) and amikacin (67.71%) had been reported highly active
against E. coli isolated from broiler chicken. Although enrofloxacin was found to be more superior in the
antibiotic activity than that of oxytetracycline and sulfadiment for control of morbidity and mortality caused
by E. coli (Saha et al., 2003). In vitro antimicrobial testing of the isolated E. coli had been reported that 14
antimicrobial drugs revealed that 90% of the isolates of E. coli were sensitive to gentamycin and gentadox.
They recorded that the organism was moderate sensitive (30-65%) to amoxicillin,
sulfamethoxazole/trimethoprim, doxystin, chloramphenicol, enrofloxacin, furozolidine, norfloxacin,
neomycin and ciprofloxacin. The findings of current research demonstrated that quinolones group;
ciprofloxacin, enroflaxacin and ofloxacin effective against the isolated organism. Whereas, cephalosporin
group; cefipime, ceftazidime and cefoxitin showed good efficacy against the organism.
Journal of Rural Development and Agriculture (2017) 2(1): 66-71
69
Table 1 The mean sensitivity of E. coli against the various antibiotics observed during study
S. No.
Antibiotic used
Code
Zone of inhibition (mm)
A
Quinolones
1.
Ciprofloxacin
CIP5
14
2
Ofloxacin
OFX5
11
3
Rifampicin
RD5
02
4
Lincomycin
MY10
R
5
Bacitracin
B10
R
6
Enrofoxacin
ENR5
12
7
Nalidixic acid
NA30
07
8
Norfloxacin
NOR10
11
B
Cephalosporins
9
Cephradin
CE30
06
10
Cefuroxime sodium
CXM30
07
11
Cefipime
FEP30
11
12
Cephalexin
CL30
5.6
13
Ceftazidime
CAZ30
11
14
Cefoxitin
FOX30
11
R- Resistant i.e. no zone of inhibition
Table 2 The minimum inhibitory concentration of different antibiotic against E. coli
Concentration of antibiotics (µg per ml media)
Antibiotic
0.8
1.6
3.2
6.4
12.5
25
50
100
Control
Ciprofloxacin
-
-
-
-
-
-
-
-
+
Metronidazole
+
+
+
+
+
+
+
+
+
Cefipime
+
-
-
-
-
-
-
-
+
Amikacin
+
+
-
-
-
-
-
-
+
+ = Growth, - = No growth
The minimum inhibitory concentration (MIC) of different antibiotics against E. coli was performed by
serially diluting antibiotics such as ciprofloxacin, metronidazole and cefipime as 0.4-50 µg/ml (Table 2). MIC
results indicated that ciprofloxacin was found to be effective to inhibit the growth of E. coli. While the MIC of
cefepime was found to be elevated concentrations of 0.4 µg/ml and 0.8 µg/ml of E. coli. In this study,
ciprofloxacin showed better efficacy than that of cefepime against the isolated E. coli. Shuyu Wu et al. (2008)
investigated in their conducted research on E. coli and found plate showed 74% effective for all strains and
agreed within ± 1 log2 dilution when comparing MICs with Mueller-Hinton II media. Whereas, they noted
significant variations for oxytetracycline and sulfamethoxazole against the organism. The description
regarding minimum inhibitory concentrations of E. coli are in accordance with the previous reports (Saha et
al., 2003; Shareef, 2004; Shuvu et al., 2008). Our findings demonstrated that the organism was highly
sensitive to ciprofloxacin.
CONCLUSION
The antibiotics of quinolones group; ciprofloxacin, enrofloxacin and ofloxacin were effective to isolate E. coli.
Whereas drugs belonged to cephalosporin group; cefipime, ceftazidime and cefoxitin showed good efficacy
against the organism. The similar MIC results were obtained for antibiotic of quinolones and cephalosporin.
The antibiogram of E. coli indicated that ciprofloxacin found to be effective antibiotic of quinolones and
cephalosporin groups.
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Author Contribution Statement Ranjhan Ali Lakho and Shahid Hussain Abro contributed in study conception and
design. Ranjhan Ali Lakho, Shahid Hussain Abro and Muhammad Tarique Tunio contributed in acquisition of data.
Mohsina Zubair, Rani Abro, Rahmatullah Rind contributed in analysis and interpretation of data. Riaz Ahmed Leghari and
Kanwar Kumar Malhi contributed in drafting the manuscript. Ranjhan Ali Lakho, Muhammad Rafique Rind, and Asghar Ali
Kamboh contributed in critical revision.
Conflict of Interest There is no conflict of interest
Acknowledgements Authors are highly thankful to Vaccine Production Unit, Tandojam, Pakistan and Central Veterinary
Diagnostic Laboratory, Tandojam, Pakistan for providing research facilities.
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