ACCRA TECHNICAL UNIVERSITY

SCHOOL OF APPLIED SCIENCES

DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY

TOPIC:

ANTIBIOGRAM OF BACTERIA ISOLATED FROM POULTRY BIRDS FROM AGNES

POULTRY AT NUNGUA ESTATE

RESEARCH PROPOSAL

BY

AGBALENYO ESINAM JOYCE

(01161818D)

Supervisor…………………………………………………………………………………….

SIGNATURE……………………. DATE………………….

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 INTRODUCTION

Poultry can be defined as domestic fowls including chickens, turkey, geese and ducks raised for

the production of meat and eggs (The American Heritage Dictionary of the English language

,2009). The world is used for flesh of these birds as food (Food and Agriculture Organization

,2012). Poultry rearing is considered superior to other agricultural sector because of the quick

economic return in relatively short period. These a strong evidence that the use of antimicrobial

agents can lead to the emergence and dissemination of resistant E. coli by Vander and

stobberingh ,2001; Schroeder, Zhao, Debroy &Toacolini ,2002) which can then be passed out to

people via food or through direct contact with animals. The finding of multiple resistant

commensal E. coli isolated from poultry birds. E. coli is a member of the family

Enterobacteriaceae, which includes many known pathogens such as salmonella shigella and

Yesinia.

Bacteria such as Escherichia coli 0157:H7 can cause severe abdominal cramps, bloody diarrhea

and vomiting coli bacillosis infections caused by the bacterium, E coli can cause infection under

the skin known as cellutis and is commonly associated with respiratory disease in birds which in

severer cases leads to septicemia and death. Although most stains of E. coli are not regarded as

pathogens theses can be in immune compromised host. Most strains of E. coli live as

commensals as opportunistic pathogens of humans and animals (Levine, 1983) in poultry,

especially in poultry birds.

Antibiogram is a result of laboratory testing for the sensitivity of an isolated bacteria strains to

different antibiotics or is an overall profile of antimicrobial susceptibility testing results of a

specific microorganism to a battery of antimicrobial drugs. This profile is generated by

laboratory using appropriate data from a hospital’s or health care system. Data are summarized

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periodically and presented showing percentage of organisms tested that are susceptible to

particular antimicrobial drugs that are regularly tested. Antibiogram helps and guide clinician

and pharmacist in selecting the best empirical antimicrobial treatment in the vent of pending

microbiology culture and susceptibility results. They are also useful tool for detecting trends in

antimicrobial resistance. When antimicrobial susceptibility testing data are summarized

cumulatively for a hospital, healthcare system or other health care facility periodically trends in

resistance and can be identified and investigated (National Committee for Clinical Laboratory

Stands, 2002.) it has been observed that antibiotics susceptibility of bacterial isolate is not

constant but dynamic and varies with time and environment(Hassan, 1995) this therefore

demands the need for period screening of common bacteria pathogen for their antibiotic

susceptibity profile. According to Aibini, (2004) E. coli is highly resistant to ampicillin

amoxicillin, tetracycline, trimethoprimsulfamethoxale. The wide spread occurrence of drug

resistant. E.cole and other pathogen in our environment has made it necessary for regular

monitoring of antibiotic susceptibility trends to provide the basis for developing rational

prescription programs making policy decision and assessing the effectiveness(Omigie, Enweani,

Ohenhen, Omohu and Osagie, 2006).

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 PROBLEM STATEMENT AND JUSTIFICATION

According to Hassan, 1995 antibiotics susceptibility of bacterial isolates is not constant but

dynamic and varies with time and environment. when this happen the microorganisms such as

salmonella spp, E.coli are sensitive to antibiotic such as amoxicillin, tetracycline, ampicillin and

etc. these microorganism affects the poultry birds and causes diseases such as colibacillosis etc.

Research shows that, when human are exposed to sick poultry birds their diseases or illness can

be transmitted to humans.

AIM

To investigate the antibiogram of bacteria isolated from poultry birds.

specific objectives

 To collect swabs from the cloacae of the poultry birds.

 To isolates bacteria from swabs by culturing

 To identify isolates by gram staining and biochemical test

 To perform antimicrobial susceptibility test on the isolates using the Kirby-Bauer method

SIGNIFICANCE OF STUDY

This study will help clinicians and pharmacist in selecting the best empiric antimicrobial

treatment and hence attention will be paid for personnel hygiene in processing products and

excess use or abuse of antibiotics will be reduced or stopped by the judicious application of

antibiotics for the safety of public health.

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 RESEARCH DESIGN AND METHODOLOGY

MATERIALS AND EQUIPMENTS

 Swab sticks

 Agar media

 Ice chest

 Glass petri dish

 Autoclave

 Bunsen burner

 Antimicrobial agent

 Peptone broth

 Inoculating loop

 Forceps

 Microscope

 Heating mantle

 Sterilizer

 Laminar airflow cabinet

METHODS

Population and study sample

Samples will be picked randomly at Nungua estate poultry farm.

Farm audit

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A structured questionnaire will be used to conduct an audit of the conditions in the poultry farm.

Sample size and selection of sample

Sterile cotton swabs will be used to collect cloacae swab from ten (10) randomly selected live

birds. This will be broken into a containing peptone water and transported to the lab.

Isolation and Identification

The broth cultures will be incubated overnight at 35°C. All the broth culture will be sub cultured

on MacConkey agar plates and nutrients agar plants will be incubated. The plates will be

incubated overnight at 35°C and examined for presence of colonies isolates on Nutrient agar of

different morphologies will be gram stain isolates showing different colony morphology on

MacConkey will be characterized by the following bio chemical identification test, catalase,

oxidase, citrate, indole urea and kliglers reaction test.

Gram staining

Air -dried, heat-fixed smear of cells will be flooded with crystal violet staining reagent for

1minute.it will be ensured that the quality of the smear (cell concentration) will not be too heavy

or too light as it will affect the gram stain results. The slide will be washed in a gentle and

indirect stream of tap water for 2 seconds and it will be flooded with the mordant: Gram’s iodine

and left to stand for 1 minutes. The slide will be washed in gentle and in direct stream of tap

water for 2 seconds and then it will be flooded with decolorizing agent and left for 15 seconds or

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drop of decolorizing agent will be added to slide until the decolorizing agent running from the

slide runs clear. The slide will later be flooded with counterstain, safranin and left to dry for

about 30 seconds to 1 minute. Finally, the slide will be washed in a gentle and indirect stream of

tap water until no color appears in the effluent and then it will be blotted dry with absorbent

paper. The result of the staining procedure will be observed under oil immersion using a

brightfield microscope. At the completion of the Gram stain, gram negative bacteria will stain

pink/red and gram-positive bacteria will stain blue/purple (Smith & hussy,2005).

Catalase test

A clean glass slide will be divided into two sections using grease pencil one will be labelled as

‘test’ and the other as ‘control’. Small drop saline will be placed on each area of the slide. A

sterilized and cooled inoculating loop will be used to pick up a small amount of the culture from

the nutrient agar petri plate. One or two colonies will be emulsified on each drop to make a

smooth suspension. A Pasteur pipette will be used to a drop of hydrogen peroxide over the test

smear. Immediately bubble formation will be observed indicating a positive result

(Prescott,Harley&klein,n.d.).

Oxidase test

Cloaca sample will be dip into the reagent and then touch colony. A blue color will be examined

after 10 seconds indicating a positive result (Snell, Brown, & Robert, 1999).

Citrate test

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The slant will be streaked back and forth a light inoculum picked from the centre of well isolated

colony. The slant will be incubated periodically at 35 degree Celsius up to 4 to 7 days. A color

change will be observed from green to blue along slides, indicating a positive result

(MacWilliams,2009).

Urease test

Slope will be inoculated heavily over the surface of the agar plate. Inoculated slope will be

incubated at 35-37˚C in water bath in an incubator slope will be examined after 4 hrs and after

overnight incubation. Purple or pink color will be observed indicating a positive results and color

of media remains unchanged indicating a negative result (Lippincott &Wilkins ,2000)

Kligler's test

To inoculate carefully touch the centre of an isolated colony on an enteric plated medium with a

cool sterile needle, stab into the medium in the butt of the tube and then streak back and forth

along the surface of the slant. Several colonies from each primary plate should be studies

separately, since mixed infections may occur. Incubate tubes with loosened caps for 18-24hrs at

35̊C in an aerobic atmosphere. Tubes will be examined after 18-24 hours for growth and

reactions (Ewing, 1986).

Indole test

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Inoculate the tryptophan (peptone broth) with the test organism and incubate at 35˚C for 25-48

hrs add 0.5ml of the kovac reagent and examine the upper layer of liquid for positive result red

color appears, yellow color appears for a negative result (Miller & Wright,1982).

Antimicrobial Susceptibility

Testing using Kirby Bauer disk diffusion method following the clinical and laboratory standards

institute, CLSI (CLSI, 2006) guidelines will be performed using a sterile wire loop. 3-5 well-

isolated color of similar appearance will be packed and emulsified in 3-4 ml of peptone water to

obtain a bacterial suspension of 0.5 McFarland turbidity standard. A sterile swab will be dipped

into the inoculum and excess fluid drained by pressing and rotating the swab against the inside of

the tube above level of the bacteria suspension.

The inoculums will be swabbed on the surface of Mueller-Hinton agar plate in three directions

rotating the plate 180° c after culture to ensure even distribution. The plate will be covered and

the surface of the agar will be allowed to dry for 3-5 minutes. Using sterile forceps, gram

positive and gram negative antimicrobial multidisc will be placed on the agar surface of their

corresponding plates and tapped lightly to ensure proper contact. Within 15 minutes of applying

the disc, the plates will one of be inverted and incubated for 16-20 in ambient air. After

overnight incubation the diameter of each zone of inhibition will be measured from the back side

of the plate using a ruler. Using antimicrobial susceptibility testing breakpoint tables from CLSI

isolates will be classified as resistant, intermediate or sensitive to each of the antibacterial agents

on the panel of multidisc. A control test will be set up using Escherichia coli: ATCC 2592

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 DATA ANALYSIS

All laboratory data will be captured directly into the log book and entered into Microscoft office

excel and imported into statistical package for social scientist 20.0 for analysis. Antibiogram of

isolates will be described using bar graph. Frequency table will be used to describe the

prevalence of isolates.

EXPECTED OUTCOME

REFERENCES

Aednipekun, E., Aibinu, I., & Odugbemi, T. (2004). Emergence of quinolone resistance amongst
Escherichia coli strain isolated from clinical infections in some lagos states. Journal of
health biomedical sciences, 3, 73-78.
Ewing. (1986). Edwards and Ewing's identification of the enterobacteriaceae (4th ed.). New
york: Elsevier science publishing company,Inc.

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Hassan, S. H. (1995). Sensitiviy of salmonella and shigella to antibiotics and chemotherapy in
sudan. Journal of tropical medicine hygiene, 88, 243-248.
Levine, M. M. (1983). Escherichia coli that cause
diarrhoea:Enterotoxigenic,enteroinvasive,enterohemorrhagic and enteradherent. Journal
infections, 155, 119-121.
Lippincott, W., & Wilkins. (2000). Biochemical tests for idetification of medical bacteria (3rd
ed.). (J. MacFaddin, Ed.)
MacWilliams, M. P. (2009). Citrate test. Retrieved from
https://www.microbiology.org/component/resource/laboratory test/3203-test-protocol
Miller, J., & Wright, J. (1982). Spot indoles test. Journal of clinical microbiology, 15, 589-592.
National committee for clinical laboratory stands. (2002). Analysis and presentation of
cummulative antimicrobial suspectibility test data.
Ochei, J., & Kolhatkar, A. (2000). Identification methods. In Medical laboratory science theory
and practice (pp. 78-97).
Omigie, O., Enweani, I. B., Ohenhen, R. E., Umohu, I. P., & Osagie, B. O. (2006).
Bacteriological survey of wound infections. Benin city, nigeria.
Schroeder, C. M., Zhao, C., Debroy, C., & Toacolini, J. (2002). Antimicrobial resistance of
Escherichia coli Ol57:H7 isolated from humans,cattle,swine and food. Journal of applied
environmental microbiogy, 68, 578-581.
Snell, J., Brown, D., & Roberts, C. (1999). Quality assurance principles and practice in the
microbiology laboratory. London.
Vanden, A. E., & Stobberingh, E. E. (1999). Antibiotic usage in animals. Journal of impact on
bacterial resistance and public health drugs, 58, 589-607.

Appendices

Questionaires

Name of farm

Location of

poultry farm

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Type of poultry

birds

Breeds of poultry

birds

Ages of the birds

Names of

antibiotics

administered

Frequency of

antibiotics

administration

Codes of

collected swabs

BUDGETS

ITEM NUMBER DESCRIPTION UNIT COST TOTAL TOTAL COST

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 NUMBER

1 Petri dish 4.00 10 40.00

2 B;juole bottle 5.00 10 5.00

3 Ruler 1.50 1.50p

4 Transportation 33.00

5 MacConkey agar 270.00

6 Nutrient agar 270.00

7 Swab sticks 40.00 1 pack 40.00

8 Peptone water 256.00

9 Mueller –Hinton 400.00

10 Slides and cover

ship

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 JUSTIFICATION

 Swab sticks will be used to collect the swab sample.

 Mueller –hinton will be used to test for antimicrobial susceptibility.

 Urea broth will be used to test for the presence of urease producing organisms.

 Peptone water will be used to detect indole and carbohydrate fermentation pattern of

non-fastidious organisms.

 Sabouraud dextrose agar will be used to isolate some selected organism (dermaphyte

Simmons citrate agar will be used to identify the ability of organisms to utilize citrate as a

carbon carbon source.

 Nutrient agar will be used to cultivate a wide range of microorganisms.

 MacConkey agar will be used to isolate and differentiate enteric based on their ability to

ferment lactose.

TIMELINE

Activity Time(weekly)
1 2 3 4 5

Sample preparation and preparation

Specific Objective 1

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Specific Objective 2

Specific Objective 3

Data Analysis and Report Submission

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