Antimicrobial Resistance of E. coli Isolates from Pakistani Chickens

This report presents a study on the isolation and identification of antibiotic susceptibility of E. coli isolated from broiler chickens in Pakistan, conducted at Quaid-i-Azam University. The research aims to determine the resistance patterns of E. coli strains to various antimicrobial agents, including those of veterinary and human significance. The study employed the disc diffusion method (Kirby Bauer method) to assess the in vitro antibiotic activities of 32 different antibiotics against the isolated E. coli strains. The results indicated high rates of resistance to several antibiotics, notably Nalidixic acid and Lincomycin, with varying levels of resistance observed across other antibiotics. The study highlights the impact of antibiotic use on the emergence of resistance and recommends surveillance programs to monitor antimicrobial resistance in pathogenic bacteria, emphasizing the potential for transmission of resistant clones from poultry to humans. The research provides valuable insights into the current state of antibiotic resistance in E. coli within the poultry sector and its implications for both animal and public health.

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Isolation and identification of antibiotic susceptibility
of
E-coli isolated from chicken
SYNOPSIS
Submitted By:
Ahmed Yar Khoso
Aneela bibi
Submitted To:
Dr. Shagufta Shafique
Faculty of Biological Sciences
Quaid-i-Azam University
Islamabad, Pakistan
2019

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Abstract:
Antimicrobial agents are being used extremely in order to reduce massive losses
caused by E. coli infections (colibacillosis) in Pakistani poultry sector. In this study
100 avian morbific E. coli strains will be isolated from broiler chickens
with colisepticemia and will analyze for susceptibility to antimicrobials of veterinary
and human significance. In vitro antibiotic activities of 32 antibiotic substances
against strains will be determined by disc diffusion method (Kirby Bauer
method). Multiple resistances to antibiotics WILL BE observed in all the
isolates. The highest rate of resistance will be against Nalidixic
acid (100%), Lincomycin (100%). Resistance to Gentamicin will not observe and to
Amikacin, Cefazolin, Colistin, Tobramycin, Ceftizoxime, Cefixime, Lincospectin,
Ceftazidime and Florfenicol will low.
This study will show resistance against the antibiotic is directly related to the its
use. These results will confirm a substantial increase in the incidence of
antimicrobial resistance in the E. coli strains is most probably due
to increase in antibiotics as feed additives
for growth, promotion and prevention of diseases, treatment of diseases used
in poultry.
The present study recommends introduction of surveillance programs to monitor
antimicrobial resistance in pathogenic bacteria is strongly needed because other
than animal health problems, transmission of resistant clones and resistance plasmids
of E. coli from animals i.e. poultry to humans can occur.
Keywords: Antibiotics susceptibility, Escherichia coli, chicken.
1. Introduction:
Escherichia coli is the most important agent causing secondary
bacterial infection in poultry and may also be a primary pathogen (Gross, 1994).
Colibacillosis is the most frequently reported disease in surveys of poultry diseases or
condemnations at processing (Saif, 2003). Colibacillosis refers to any localized or
systemic infection caused entirely or partly by avian pathogenic Escherichia coli
(APEC), including colisepticemia, coligranuloma (Hjarre’s disease), air sac disease
(chronic respiratory disease, CRD), cellulites (inflammatory process), swollen-head
syndrome, peritonitis, salpingitis, osteomyelitis/synovitis (turkey osteomyelitis
complex), panophthalmitis, and omphalitis/yolk sac infection (Saif, 2003).
Colisepticemia is the Well-known form colibacillosis and cause hugeeconomic losses
in aviculture throughout the world (Barnes and Gross, 1997; Ewers, et al., 2003). In
the past few years, both the incidence and severity of colibacillosis have increased
rapidly, and current trends indicate that it is probably to continue and become an even
greater problem in the poultry industry (Altekruse et al., 2002; Blanco, 1997).

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Antimicrobial therapy is an important tool in reducing both the incidence and
mortality associated with avian colibacillosis (Freed et al., 1993; Goren, 1990; Watts
et al., 1993). E. coli may be sensitive to many antibiotics. However, isolates of E. coli
from poultry are frequently resistant to one or more antibiotics, especially if they have
been widely used in poultry industry over a long period (e.g., tetracyclines) (Allan et
al., 1993; Blanco et al., 1997; Watts et al., 1993). Antibiotics once effective at
controlling E. coli infections are now ineffective due to the bacterium’s acquired
resistance to these compounds. Resistance to two or more classes of antibiotics is now
commonplace in both veterinary (Gonzalez and Blanco, 1989; Harnett and Gyles,
1984; Irwin et al., 1989) and human (Dennesen et al., 1998) medicine.
Resistance generally occurs following response to prior contact with the antimicrobial
but can occur naturally in the absence of previous exposure. Antibiotic usage is
possibly the most important factor that promotes the emergence, selection and
dissemination of antibiotic-resistant microorganisms in both veterinary and human
medicine (Neu, 1992; Witte, 1998). Concern about antibiotic resistance and its
transmission to human pathogens is important because these resistant bacteria may
colonize the human intestinal tract and may also contribute resistance genes to human
endogenous flora. Furthermore, fluoroquinolones are highly efficacious antimicrobial
agents, which because of their low toxicity and relatively broad-spectrum coverage,
are extremely valuable for treating human infections (Angulo, et al., 2000; Livermore
et al., 2002). In the past years they are used in treatment of certain bacterial diseases
in animals, including acute bovine respiratory disease and avian colibacillosis
(Hooper, 2001; White et al., 2000; Yang et al., 2004). However, a growing number of
studies report an association between the emergence of fluoroquinolone-resistant
zoonotic pathogens, such as Salmonella, E. coli, and Campylobacter, and the
subsequent approval and use of these agents in veterinary medicine (Blanco et al.,
1997; Guerra et al. 2003; Saenz et al., 2003; Yang et al., 2004). Garau et al. 1999)
suggested that the high prevalence of fluoroquinolone-resistant avian E. coli in the
stools of healthy humans in their area (Barcelona, Spain) could be linked to the high
prevalence of resistant isolates in poultry and pork (Garau et al., 1999).
In this study, E. coli strains isolated from
broiler chickens having died from colisepticemia were analyzed to determine their
susceptibilities to antimicrobial agents used in veterinary and human.
2. Materials and Methods
2.1 History:
Study papulation will be E. coli that will be isolated from 100 Commercial broiler
chicken that had died from colisepticemia, from July 2018 till October 2020. All the
concerned data will be available at Poultry research institute. Isolation and
identification of antibiotic sensitivity of E-coli isolated from chicken then will be
done by different standard bacteriological and biochemical methods.

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2.2 Bacterial isolates:
a) Bacteriological culture:
The specimens will be directly inoculated into blood agar, and the plates will be
inverted and incubated aerobically at 37°C for 24 h, after which the plates are
examined for growth( (BBL Microbiology Systems, Cockeysville, Md.)Pure cultures of
bacteria will be obtained by aseptically streaking representative colonies of different
morphological types on blood agar then we will examine the colony with naked eye
for their morphological properties and any change in the media (Gross, 1994). The
isolates will then culture on MacConkey agar and Salmonella shigella agar to
differentiate colonies of bacteria and a loop of the isolates will inoculate into nutrient
broth for further investigation. MacConkey agar will give us pink color of E. coli.
b) Grams staining:
This method will use to differentiate bacteria into Gram-negative (pink) and Gram-
positive bacteria (purple). It will base on the ability of the bacteria to retain the color
of stains used during gram reaction. Gram-negative bacteria wil bel decolorized by
alcohol, losing the purple color of the primary stains (crystal violet), but Gram-
positive bacteria will not be decolorized by alcohol and remain as purple. After
decolorization step, a counter stain (carbol fuchsine) will be used to impart a pink
color to the decolorized Gram-negative bacteria. E. coli is Gram-negative bacteria
(Clokie et al.2011; Loc-Carrillo and Abedon 2011)
c) Biochemical tests:
These are a series of tests that will be used to identify bacteria into its generic level
based on the types of enzyme produced and metabolism type performed by bacteria
with sugars. Different primary and secondary biochemical tests will be used are
Catalase, Oxidase, Motility, Oxidative-Fermentative test (O-F) and the IMViC series
that consist of four definitive tests: indoleproduction, the methyl red test, the Voges-
Proskauer test, and the citrate utilization test Wang (Clark and Rodgers 2002). For the
isolation of E. coli like colonies (colonies with no hemolysis on blood agar and
colonies with lactose fermenting on macConkey agar), Catalase (+), Oxidase (-),
Motility (+), O-F (F) tests will be used to make sure that the isolated colonies are
suggestive for E. coli after which secondary biochemical tests, IMViC will be used
to identify the isolated E. coli from other coliform bacteria (Fagan et al.1999)

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2.3 Antimicrobial susceptibility determination:
Antimicrobial susceptibility determination will be routinely tested by the single-disc
diffusion method (Bauer et al. [Am. J. clin. Path. 45, 493-496 (1966)]. The E. coli
strains will test against the antibiotics of human and veterinary significance. The
following antibiotic discs on Mueller Hinton agar will be applied:Amikacin (AN/30
μg), Ampicillin (AM/10 μg), Amoxicillin (AMX/30 μg), Cefazolin (CZ/30 μg),
Cefixime (CFM/5 μg), Ceftazidime (CAZ/30 μg), Ceftizoxime (CT/30 μg),
Chloramphenicol (C/30 μg), Chlortetracycline (CTe/30μg), Ciprofloxacin (CP/5 μg),
Colistin (Cl/10 μg), Difloxacin (DIF/25 μg), Doxycycline (D/30 μg), Enrofloxacin
(NFX/5 μg), Erythromycin (E/15 μg), Florfenicol (FFc/30 μg), Flumequine (Flu/30
μg), Furazolidone (FR/100 μg), Gentamicin (GM/10 μg), Kanamycin (K/30 μg),
Lincomycin (L/2 μg), Lincospectin (LIN/SE), Nalidixic acid (NA/30 μg), Neomycin
(N/30 μg), Nitrofurantoin (FM/300 μg), Norfloxacin (NOR/10 μg), Oxytetracycline
(T/30 μg), Streptomycin (S/10 μg), Tetracycline (TE/30 μg), Tiamulin (TM/ 30μg),
Tobramycin (TOB/10 μg) and Trimethoprim-Sulphamethoxazole (SXT/25 μg).
2.4Plan B
If we will not get the expected result, then alternative antibiotic will be used.
2.5 Timeline
Total time for my research work is two years; I will carry out my research work as per
schedule to avoid any trouble shooting.

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3. Expected Results
The highest rate of resistance was against Nalidixic acid (100%), Lincomycin (100%),
Erythromycin (97%), Oxytetracycline (95%), Chlortetracycline (95%), Tetracycline
(94%), Flumequine (94%), Tiamulin (91%), Doxycycline (88%), Difloxacin (83%),
Neomycin (81%), Streptomycin (81%), Trimethoprim-Sulphamethoxazole (80%),
Kanamycin (77%), Enrofloxacin (76%), Norfloxacin (68%), Ciprofloxacin (67%),
Chloramphenicol (67%), Furazolidone (66%), Nitrofurantoin (56%), Amoxicillin
(53%), and Ampicillin (47%). Low levels of resistance were against Florfenicol
(27%), Ceftazidime (18%), Lincospectin (15%), Cefixime (14%), Ceftizoxime (7%),
Tobramycin (7%), Colistin (6%), Cefazolin (4%), Amikacin (3%), and Gentamicin
(0%).
4. Conclusion
Increase use of antibiotics for health and economic purpose will enhance the
incidence of resistance against antibioics in the Ecoli starins.

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