development of antibiotic detection bioassay kit from ... · ii abstract background: the presence...

Islamic University of Gaza

Deanship of Research and Graduate Studies

Faculty of Science

Master of biotechnology

زةةةةةةةةةةةةةةةةةةةةةةةةةةةةغب الجامعةةةةةةةةةةةةةةةةةةة ا ةةةةةةةةةةةةةةةةةةة م

ةةةةةامالبحةةةةةل العممةةةةةت اال ا ةةةةةا الع عمةةةةةا ة

العمةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةة م ةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةةك

ماجسةةةةةةةةةةةةةةةةا ح الا ل ج ةةةةةةةةةةةةةةةةا الح ةةةةةةةةةةةةةةةة

Development of Antibiotic Detection Bioassay Kit from

Locally Isolated Bacteria

تطىير فحص حيىي للكشف عن متبقيات المضادات الحيىية باستخذام بكتيريا

محليامعزولة

By

Ayat A. Elkurd

Supervisor by

Dr. Abdelraouf A. Elmanama Dr. Kamal J. Elnabris

Marine biologyof Assoc. Prof. . of MicrobiologyProf

A thesis submitted in partial fulfillment

of the requirements for the degree of

Biotechnology Master of

November 2017

I

إقةةةةةةةحا

أنا الم قع أ ناه مق الح ال الات تحمل ع ان:

Development of Antibiotic Detection Bioassay Kit

from Locally Isolated Bacteria

المضادات الحيىية تطىير فحص حيىي للكشف عن متبقيات

باستخذام بكتيريا معزولة محليا

ألش بأ يب اشخهج عه ز انشسبنت إب خبج خذ انخبص، ببسخثبء يب حج اإلشبسة إن

حثب سد، أ ز انشسبنت ككم أ أ خزء يب نى مذو ي لبم اخش نم دسخت أ نمب

.أ بحثت أخش عه أ بحث نذ أ يؤسست حعهت

Declaration

I understand the nature of plagiarism، and I am aware of the University’s

policy on this. The work provided in this thesis، unless otherwise referenced،

is the researcher's own work، and has not been submitted by others elsewhere

for any other degree or qualification.

آيا ال ح اسم الطالب:Student's name:

:Signature التوقيع:

:Date التاريخ:

II

Abstract

Background: The presence of antibiotic residues in milk may cause different

diseases or disorders like, direct toxic effects, allergic reactions in individuals with

hypersensitivity, and can result in the development of resistant strains of bacteria in

consumers. In addition, these residues can modify or inhibit the fermentation

processes performed in dairy products such as cheese and yoghurt. The

determination of antibiotic residues in raw milk is usually performed by two

methods. Microbial and enzymatic methods, like microbial growth inhibition tests.

Tests utilize bacterial test strains such as Bacillus stearothermophilus var.

calidolactis, Streptococcus thermophilus and Bacillus subtilis ATCC 6633.

Objective: The main objective of this study is to develop antibiotic residues

detection bioassay kit from locally isolated bacteria from soil sample in Gaza strip.

Methods: soil samples were collected from different locations in the three governorates at

Gaza strip.116 bacteria isolates were isolated from soil sample. The bacteria isolates were

identified as the most resistance of all antibiotics. They allowed to grow in different cultures

at 60 ° C. After that, a suitable the culture media developed for growing of the isolates

bacteria. Collecting 81 milk samples and then determination the containing antibiotic

residues using a commercial test (Mira test) and the locally developed test .

Results: . The percentage of presumably positive results after 4 hours experiment was 82.7,

79.0 and 76.5 for the LDMBB-4 h, LDMBG-4 h and MiRA Test-4 h respectively. After 24

h, this percentages were dropped to 49.4, 35.8 and 17.3% in LDMBB-24 h, LDMBG-24 h,

and MiRA Test-24h respectively. Results of the chi-square test of homogeneity revealed a

statistically significant difference (p < 0.001) in proportions of positive (or negative) residues

in milk samples between the six experimental trials.

Conclusions: The result was statistically significant compared to locally kit and commercial

kit. Therefore, we recommend that used for locally kit, when to use it positive effects on

consumer health

Key words: raw milk, antibiotic resistance, residual antibiotic, develop local kit,

commercial kit

III

ملخص الرسالة

ا خد يخبمبث انضبداث انحت ف انحهب لذ سبب ايشاض اخطبس يخخهفت يثم اثبس انسبيت المقذمة :

انببششة انحسبست ف األفشاد انز عب ي فشط انحسبست، ك ا خح سالالث يمبيت ي انبكخشب

ك ا حعذل ا حثبظ عهبث انخخش إلخبج يخدبث االنبب ف انسخهك. ببإلضبفت ان ا ز انخبمبث

انخخهفت يثم اندب انهب. ححذذ يخبمبث انضبداث انحت ف انحهب انخبو خى بطشمخ طشلت اسخخذاو

انكشببث أ االزبث يثم حثبظ انكشببث .سخخذو نز االخخببساث بكخشب ي االيثهت عهب

Bacillus stearothermophilus var. calidolactis, Streptococcus thermophilus and

Bacillus subtilis ATCC 6633.

ي ز انذاسست حطش فحض ح نهكشف ع يخبمبث انضبداث انحت ببسخخذاو : انذف انشئس الهذف

بكخشب يعزنت ي عبث حشبت ي لطبع غزة .

ى ححذذ ث ،عزنت بكخشب 116. حى عزل يبطك يخخهفت ف لطبع غزة 5عت حشبت ي 92حى خع الطرق :

انعزنت االلذس عه يكبفحت خع انضبداث انحت ثى االلذس عه ان بأسبط غزائت يخخهفت عه حشاسة

إيكبت عت حهب ثى ححذذ ١٨، خع سظ غزائ يبسب ن انبكخشب دسخت يئت ، بعذ رنك حى حصى٠٦

عه يخبمبث انضبداث انحت ببسخخذاو فحض حدبس )فحض يشا ( انفحض انطس يحهب احخائب

. ببنذساست

LDMBB-نالخخببساث%76.5 82.779.0سبعبث ي انخدشبت 4كبج انسبت انحخهت نهخبئح بعذ : النتائج

4 h LDMBG-4 h MiRA Test-4 49.4 سبعت ي انخدشبت كبج انسبت 94عه انخان .بعذ

عه انخان .عذ h-LDMBB 24 h-LDMBG 24 h-MiRA Test 24نالخخببساث %17.3 35.8

p < 0.001( نكشف انفشق ر انذالالث االحصبئت كبج انخدت (chi-square testحطبك اخخببس انخدبس

. ال خذ فشق ر دالنت احصبئتانسبنبت نخبمبث انضبداث انحت ف عبث انحهب. أنهسب انخبت ا

باستخدامالتجاري لذا نوص الفحصمحل الصنع و الفحصكانت النتجة ذو داللة احصائة بالمقارنة بن الخالصة :

.ستخدام من ااار اجابة للى صحة المستلل ال محلى الصنع لما الفحص

انطس انفحض ،يخبمبث انضبداث انحت يمبيت انضبداث انحت، ،انحهب انخبوالكلمات المفتاحية:

انخدبس . انفحضيحهب ،

IV

Dedication

I dedicate this work to:

The soul of my dad ----the greatest man ever

My mother who spent her life seeking our comfort and happiness.

My dear husband who helped me during my education journey.

All who had a role in finishing this research.

Great professors:

Prof. Dr. Abdelraouf A. Elmanama

Assoc. Prof. Dr. Kamal J. Elnabris

To my dear sister who supported me.

V

ACKNOWLEDGEMENTS

"Who he doesn't thank people doesn't thank Allah"

My dear Mum; the greatest woman ever: I would love to thank you for all you had

given.

My husband; I thank you for supporting me in my education journey.

Prof. Dr. Abdelraouf A. Elmanama and Assoc. Prof. Dr. My great teachers,

I would like thank you all for all you had taught us Mr. Kamal J. Elnabris

Mohamed albayome , Miss. Mareame alrefy, Miss. Alaa marouf and Miss.

Karama Nasar thank you.

I dedicate you all this work and would love to thank you for helping in bringing this

work to light.

VI

Table of Content

I .......................................................................................................................... إلـــــــشاس

Abstract ........................................................................................................................ II

III ................................................................................................................. يهخض انشسبنت

Dedication .................................................................................................................. IV

ACKNOWLEDGEMENTS ......................................................................................... V

Table of Content ........................................................................................................ VI

List of Tables ............................................................................................................. IX

List of Figures ............................................................................................................ XI

List of Abbreviations ................................................................................................ XII

Chapter I ...................................................................................................................... 2

Introduction .................................................................................................................. 2

Overview1.1 ............................................................................................................. 2

1.2 Objectives …………………………………………………………………….5

1.2.1 General objective ...................................................................................... 5

1.2.2 Specific objectives .................................................................................... 5

1.3 Significance ...................................................................................................... 5

Chapter II ..................................................................................................................... 8

Literature review .......................................................................................................... 8

2.1 Effect of antibiotic usage in cows .................................................................... 8

2.2 Withholding period ............................................................................................ 9

2.3 Adverse effects of using antibiotic in milk ........................................................ 9

2.4 The Genus Bacillus .......................................................................................... 11

2.4.1 Characters of bacillus species ....................................................................... 11

2.4.2 Bacillus Species: ........................................................................................... 12

2.4.3 B. cereus group and B. subtilis group ........................................................... 12

2.4.4 Bacillus used of production many enzymes ................................................. 13

2.5 Detection methods of antibiotic residues in milk ............................................. 13

2.5.1 Bioassay ................................................................................................. 13

2.5.1.1 Definition ............................................................................................ 13

2.5.1.2 Principle of Bioassay ........................................................................... 14

2.5.1.3 Delvotest ............................................................................................. 14

VII

2.5.1.4 Penzyme test ........................................................................................ 15

2.5.1.5 Copan test ............................................................................................ 16

2.5.1.6 Charm Farm test .................................................................................. 17

2.5.1.7 Idexx parallux (antibiotic test kit) ........................................................ 18

2.5.1.8 Microbiological system in microtitre plates ......................................... 19

2.5.2 ELISA .................................................................................................... 19

2.5.3 Chromatography ..................................................................................... 20

2.5.4 HPLC-MS/MS method ........................................................................... 21

Chapter III .................................................................................................................. 23

Materials and Methods ............................................................................................... 23

3.1 Materials ........................................................................................................... 23

3.1.1 Apparatus ............................................................................................... 23

3.1.2 Chemicals, culture media and antibiotics ................................................ 23

3.1.3 Microorganism maintenance and storage ................................................ 24

3.2 Methodology ................................................................................................... 24

3.2.1 Soil Sample collection ............................................................................ 24

3.2.2 Isolation of Bacillus spp. ......................................................................... 25

3.2.3 Antibiotic susceptibility by agar diffusion method ................................. 25

3.2.4 Thermal resistance studies and identification .......................................... 26

3.2.5 Test Media formulation .......................................................................... 27

3.2.6 Determination of antimicrobial residues and microbial load in milk

samples ........................................................................................................... 28

3.2.7 Statistical analysis .................................................................................. 30

Chapter IV .................................................................................................................. 32

Results and Discussion .............................................................................................. 32

4.1 Isolation of test microorganism ........................................................................ 32

4.2 Determination of the most antibiotic-sensitive strain ...................................... 32

4.3 Determination of the best temperature-resistant isolate ................................... 33

4.4 Characterization of isolate 25.5 ........................................................................ 34

4.5 Antibiotic susceptibility test of the Bacillus subtilis isolate ............................ 37

4.6 Media formulation ............................................................................................ 39

4.7 Microbial quality of milk ................................................................................. 40

VIII

4.7.1 Microbial quality of milk based on TBC ................................................. 40

4.8 Milk testing for antibiotic residues, test performance characteristics and residue

prevalence .............................................................................................................. 43

4.9 Performance of B. subtilis vs. G. stearthermophillus on agar medium ........... 50

Chapter V ................................................................................................................... 54

Conclusions and Recommendations .......................................................................... 54

5.1 Conclusions ...................................................................................................... 54

5.2 Recommendations ............................................................................................ 55

Reference ................................................................................................................... 57

Appendices ................................................................................................................. 61

Appendix 1: Antibiotic susceptibility of strains isolated from soil sample ........... 61

IX

List of Tables

Table (3.1): List of the apparatus used in this work .................................................. 23

Table (3.2): Media used for isolation, cultivation, identification and kit development

................................................................................................................................... 23

Table (3.3): Antimicrobial discs which was be used in this study ............................ 24

Table (3.4): Locations for the collected soil samples ............................................... 25

Table (3.5): Various media formulation which was used in this study ..................... 27

Table (3.6): Distribution of milk samples by governorates ....................................... 28

Table (4.1): Number of soil samples and isolates obtained from the four governorates

at Gaza strip ............................................................................................................... 32

Table(4.2): Growth evaluation of the 34 isolates on different media at 60oC ........... 34

Table (4.3): Phenotypic characteristics of the selected isolate (25.5) ....................... 35

Table (4.4): The antibiotic sensitivity of B. subtilis isolate ...................................... 38

Table (4.5): The basic statistical measurements for the number of micro-organisms

per milliliter of 81 tested samples .............................................................................. 41

Table (4.6) : Statistical differences in TBC in relation to the governorates (Based on

Mann-Whitney U/Wilcoxon W test) .......................................................................... 41

Table (4.7): Microbiological quality of fresh cattle milk from different governorates

of Gaza strip as judged by legal standards of Palestinian Dairy Products Regulations

................................................................................................................................... 42

Table (4.8): Results (expressed as number of positive and negative) of analysis of

milk samples by using the six experimental trials ..................................................... 44

Table (4.9): Numbers and percentages (%) of positive (+) and negative (-) results of

commercial test (MiRA Test-4 h) and locally developed medium broth with G.

stearthermophillus (LDMBG-4 h), and commercial test and locally developed

medium broth with B. subtilis (LDMBB-4 h)............................................................ 47

Table (4.10): Kappa statistics of agreement between MIRA Test and other locally

developed and modified tests and its interpretation according to the Landis Koch

scale ........................................................................................................................... 48

Table (4.11): Comparative costing for conducting 50 tests by MiRA and LDMBB

tests ............................................................................................................................ 49

X

Table (4.12): Frequencies and percentages (%) of zones of inhibition occurred at agar

plates made by locally developed medium by using B. subtilis and G.

stearthermophillus after 24 h incubation period ........................................................ 50

XI

List of Figures

Figure (21): Enzyme milk test ................................................................................... 15

Figure (2.2): Results of Penzyme milk test ................................................................ 16

Figure (2.3): Copan MICROPLATE ........................................................................ 17

Figure (2.4): COPAN MILK TEST results ............................................................... 17

Figure (2.5): Idexx parallux (antibiotic test kit) ........................................................ 19

Figure (4.1): Antibiotic sensitivity patterns among the isolated strains .................... 33

Figure (4.2): The number of milk samples collected from the four governorates at

Gaza strip ................................................................................................................... 40

Figure (4.3): Overall proportion of presumably of positive and negative results (%)

across the experimental groups after 4 h test period .................................................. 43

Figure (4.4): Overall proportion of presumably of positive and negative results (%)

across the experimental groups after 24 h test period ................................................ 44

Figure (4.5): MIRA test results after 4 h incubation period (purple color indicates

positive result and yellow color indicates negative result) ........................................ 45

Figure (4.6): LDMBB test (A) and MiRA Test (B) ................................................... 49

Figure (4.7): Solid MRVP media showing zone of inhibition ................................... 51

XII

List of Abbreviations

Maximum Residue Limits (MRLs)

Food and Drug Administration(FDA)

World Health Organization (WHO)

Food and Agricultural organization (FAO)

Bacillus stearothermophilus var. calidolactis disc assay (BsDA)

Mueller-Hinton agar (MHA)

Nutrient Broth (NB)

Brain Heart Infusion Broth (BHIB)

Methyl Red, Voges-Proskauer (MR-VP)

modified MR-VP (mMR-VP)

Nutrient blood ager (NBA)

Bacillus subtilis (B. subtilis)

Geobacillus stearothermophilus (G. stearothermophilus)

Total Bacterial Counts (TBC)

locally developed medium broth with Bacillus subtilis (LDMBB)

locally developed medium broth with Geobacillus stearthermophillus (LDMBG)

G. stearthermophillus

Enzyme linked immunosorbent assay (ELISA)

1

Chapter I

Introduction

2

Chapter I

Introduction

Overview1.1

Shortly after the discovery of penicillin by Alexander Fleming in 1928, hundreds of

other antibiotics have appeared on the market. Since many farmers began giving

antibiotics to livestock in the late 1940s, people have been infected with strains of

bacteria that are resistant to those antibiotics (Kaya & Filazi, 2010; Link, Weber, &

Fussenegger, 2007; Sarmah, Meyer, & Boxall, 2006).

While many antibiotics are known to exist, efforts to discover new antibiotics

continue. Antibiotics are used for several purposes; in humans and animals. They are

used a treat to numerous bacterial infections causing disease, as growth promoters,

and to improves feeding efficiency (Martins-Júnior, Kussumi, Wang, & Lebre, 2007;

Sarmah et al., 2006).

The amount of antibiotics consumed by livestock worldwide is estimated to be twice

that used by humans, according to some estimates. This may lead to diseases that are

not easily treated and may be complicated, and leads to the generation of drug-

resistant bacteria (Hou et al., 2015; Trevisi et al., 2014).

Drugs are delivered to animals through feed or water, by injection, implant, drench,

paste, orally, topically, pour on, and bolus (Scherpenzeel et al., 2014). In lactating

cows, antimicrobial agents are used mostly as therapy of mastitis and other diseases

such as respiratory diseases and metritis (NaVrátiloVá, 2008).

In dairy cattle, antibiotics are used for the treatment of diseases such as: bacterial

infections, especially mastitis, diarrhea, pulmonary, enteritis, pneumonia,

endometritis, septicemia, metritis and other secondary bacterial infections. The

inevitable consequences of such treatments are the presence of antibiotic residues in

milk (Beltrán, Romero, Althaus, & Molina, 2013; Freitas, Barbosa, & Ramos, 2013;

Kaale, Chambuso, & Kitwala, 2008; Nebot et al., 2012; Scherpenzeel et al., 2014).

In Gaza strip, some studies indicated the presence of contamination by antibiotic

residues in broiler chickens and fish which increases the concern about the presence

3

of such chemicals in other foods of animal origin such as milk (Elmanama &

Albayoumi, 2016).

In Gaza strip, approximately 2500 milking cows are present, with average production

of 21 liters/day/animal (Snunu, 2017).

A large variety of food products are made from cow's milk, such as cheese, cream,

butter and yogurt. These foods are referred to as dairy products, or milk products,

and they are a major part of the modern diet. Because milk and dairy foods are

considered to be one of the main food groups important in a healthy balanced diet.

Milk is considered as very important source of calcium, milk and milk products are

also an important source of good quality protein, the B vitamins, B2 (riboflavin) and

B12, and the minerals iodine, potassium and phosphorus (Palmer, 1925).

The presence of antibiotic residues in milk may cause different diseases or disorders

like, direct toxic effects, allergic reactions in individuals with hypersensitivity, and

can result in the development of resistant strains of bacteria in consumers. In

addition, these residues can modify or inhibit the fermentation processes performed

in dairy products such as cheese and yoghurt (Freitas et al., 2013; A Junza, Amatya,

Barrón, & Barbosa, 2011; Nagel, Molina, & Althaus, 2013). For these reasons,

several control legislations, determination of the maximum residue limits (MRLs) for

the different veterinary products that may be present in food, including milk (Freitas

et al., 2013; Nagel et al., 2013).

To protect milk consumer’s health from the presence of residues of veterinary drugs,

MRLs of veterinary drugs in food have been set up in the regulation. In the European

Union, the regulatory levels or Maximum Residue Limits (EU-MRLs) are defined by

Regulation (EC) 470/2009 (European Union, 2009) and established by Commission

Regulation (EU) 37/2010 (European Union, 2010). The EU has established MRLs

for several classes of antibiotics in animal products, such as milk and edible tissues,

with the aim of minimizing risk to human health (Beltrán et al., 2013; Freitas et al.,

2013; A Junza et al., 2011; Alexandra Junza et al., 2014; NaVrátiloVá, 2008; Nebot

et al., 2012).

4

In milk, the MRL ranges are between 4 and 30 μg/kg for penicillins, 20 and 100

μg/kg for cephalosporins, and 30 and 100 μg/kg for Quinolones, and Oxytetracycline

100 μg/kg. The US Food and Drug Administration Center for Veterinary Medicine

(FDA) established Safe Levels/Tolerance of antibiotic residues in milk for the

consumer protection (FDA, 2005). Levels of up to 30 μg/kg for Oxytetracycline, 30

ng/mL for chlortetracycline. Also the Food and Agricultural organization (FAO),

World Health Organization (WHO) recommends a maximum allowable the

Maximum Residue Limits (MRLs) on residual antibiotic(Beltrán et al., 2013; Freitas

et al., 2013; A Junza et al., 2011; Alexandra Junza et al., 2014; NaVrátiloVá, 2008;

Nebot et al., 2012).

Milk quality is mainly evaluated in terms of its technological or coagulation

properties, which can be affected by the presence of antibiotic residues in milk.

Several methods have been proposed to determine antibiotic residues in milk, such as

microbiological, chromatographic, immunochemical and enzymatic or receptor-

based tests(Freitas et al., 2013; Rinken & Riik, 2006).

Microbiological and bioassay techniques are still used for antibiotic qualitative

screening purposes, mainly because of their low cost and simplicity. Most of these

tests utilize bacterial test strains such as Bacillus stearothermophilus var.

calidolactis, Streptococcus thermophiles (BsDA) and Bacillus subtilis (B. subtilis)

ATCC 6633 (Freitas et al., 2013; Kaya & Filazi, 2010). FDA evaluated and approved

16 commonly used milk screening tests, including Delvotest P and Penzyme test,

BRTAiM®, Delvotest

®, CH

®-ATK Microplate P&S (Montero, Althaus, Molina,

Berruga, & Molina, 2005; Zeng, Escobar, & Brown-Crowder, 1996).

Factors to be considered in choosing the most suitable method of residue detection

are the type of antibiotic used, expected time limitations, sensitivity and costs. The

antibiotic residue detection assay systems that are currently available use different

methods and test organisms. Microbiological assays for the detection of antibiotic

residues utilize the genus Bacillus because of its high sensitivity to the majority of

antibiotics. The BsDA disc assay is routinely used by dairy industry to screen

antibiotic residues. Delvotest SP is a multiple microbial inhibitor test usable to detect

5

antimicrobial agents such as beta lactams and sulfa compounds (Iciek, Błaszczyk, &

Papiewska, 2008).

There are many disadvantage in residues antibiotic detection methods, such as

sensitivities to certain types of antibiotics only, like this, Enzyme Linked Assay

(ELISA) and Chromatography. In addition, the need of trained personnel, the use of

special equipment such the photometric reader to interpret the results in some tests,

high cost and difficulty in implementation (Jackman, Chesham, Mitchell, & Dyer,

1990; Mokh, Jaber, Kouzayha, Budzinski, & Al Iskandarani, 2014).

Although commercially available, the costs of these kits are high and may not be

affordable for routine monitoring of large numbers of milk samples. Thus, it is

necessary to develop local and cheap kits.

1.2 Objectives

1.2.1 General objective

The main objective of this study is to develop antibiotic residues detection bioassay

kit from locally isolated bacteria from soil sample in Gaza strip.

1.2.2 Specific objectives

1- To isolate bacterial species belong to the genus Bacillus from the soil,

2- To evaluate their sensitivity to antibiotics.

3- To select those that show the highest sensitivity to most or all antibiotics.

4- To develop a suitable indicator growth media.

5- To design a microbiological inhibition bioassay and compare its

sensitivity, efficiency, cost and easiness of implementation with a

commercially available one.

6- To screen milk samples for the presence of antibiotic residues.

1.3 Significance

The misuse of antibiotic leads to marketing of contaminated products with antibiotic

residues and this may have many risks to the consumer. Thus, the safety and quality

of food must be monitored to prevent risks to humans. Developing a cheap and

affordable kit for the detection of antibiotics in milk would likely encourage all

6

concerned parties to perform routine monitoring, thus providing safer milk for direct

consumption as well as for the dairy industry which is greatly affected by the

presence of antibiotics which interfere with the fermentation processes.

7

Chapter II

Literature Review

8

Chapter II

Literature review

2.1 Effect of antibiotic usage in cows

Many antibiotics are licensed for the treatment of diseases in lactating dairy cows. A

possible unwanted consequence of this treatment is the occurrence of antibiotic

residues in milk. These residues are sometimes called bacterial inhibitory substances

because of the microbiological basis of the screening tests frequently used to detect

them (McEwen, Black, & Meek, 1992).

Residues of these antimicrobial agents in milk may cause problems in the milk

processing industry (yoghurt, cheese and other dairy products. To ensure human food

safety, Maximum Residue Limits (MRLs) have been set out for many antimicrobial

agents and different methods of analysis were developed for the swift detection of

residuals of inhibitors present in milk (Montero et al., 2005).

Almost any microbe that can opportunistically invade tissue and cause infection can

cause mastitis. However, most infections are caused by various species of

streptococci, staphylococci, and gram-negative rods, the primary sources of infection

for most pathogens may be regarded as contagious or environmental(Montero et al.,

2005).

Intramammary infections are often described as subclinical, clinical mastitis or

chronic mastitis. Clinical mastitis is an inflammatory response to infection causing

visibly abnormal milk (eg, color, fibrin clots). As the extent of the inflammation

increases, changes in the udder (swelling, heat, pain, redness) may also be

apparent. Subclinical mastitis is the presence of an infection without apparent signs

of local inflammation or systemic involvement. Although transient episodes of

abnormal milk or udder inflammation may appear, Chronic mastitis which is an

inflammatory process that has lasted for months and may continue from one lactation

to another (Hospido & Sonesson, 2005).

9

Several components in mastitic milk interfere with various antibiotic residue-

screening tests. These components include somatic cells, lactoferrin, lysozyme,

microbes, and free fatty acids (Andrew, Frobish, Paape, & Maturin, 1997).

2.2 Withholding period

Withholding periods after treatment of animals should be appropriate to guarantee

that concentrations of drug residues in edible tissues and milk do not exceed the

MRL at the time of harvesting food of animal origin (milking or slaughter)

(Knappstein, Suhren, & Walte, 2003). A milk discard time applies to female animals

that produce milk for human consumption and is the interval between the last

administration of a new animal drug and when the milk produced by the animal can

be safely consumed by humans (BEILKE & FRITZ, 2016).

Milk withholding times have been established for the purpose of providing a high

degree of assurance (provided that label instructions are followed) that milk kept for

human consumption will not contain quantities of antibiotic residues that might be

harmful to humans, i.e. above tolerance levels (MRL) (McEwen et al., 1992).

Surveys have shown that farmers sometimes forget to withhold milk from treated

cows for the proper time, but other mistakes, such as withholding of milk only from

treated quarters while placing milk from untreated quarters into the bulk tank, have

also been described. Farm management factors that have been associated with an

increased risk of residues in milk include the frequent use of part-time employees,

use of medicated feeds, use of parlor milking systems, and failure to use separate

equipment to milk treated cows (McEwen et al., 1992).

2.3 Adverse effects of using antibiotic in milk

Traditional methods of milk pasteurization reduce the quantity of bacteria present in

milk to negligible levels, but will not appreciably reduce the level of antibiotic

residues. Milk can be contaminated with fecal pathogens that exhibit resistance to

antibiotics and raw milk products have been implicated as mechanisms for

transferring fecal pathogens from farm environments to humans (Mokh et al., 2014).

10

Allergic reactions to antibiotics are well recognized and hypersensitivity to β-lactam

compounds is especially prevalent. The immunological characteristics of most other

drug classes (including macrolides, tetracyclines and aminoglycosides) makes the

development of allergic responses to minute residues unlikely, although it is

considered theoretically possible that exposure could result in clinically relevant

immunological events (Mokh et al., 2014).

Allergic reactions (dermatitis, pruritis and urticaria) of pre-sensitized individuals

caused by β-lactam residues in milk have been documented for a small number of

people (Dewdney & Edwards, 1984). Exposure to penicillin residues in milk has

been reported as a cause of chronic urticaria(Boonk & Van Ketel, 1981; Ormerod,

1987).

Individuals with a history of penicillin reaction or multiple allergen sensitivity may

experience a severe life-threatening allergic condition called anaphylactic shock

within minutes to hours of exposure to penicillin. Symptoms of severe allergic

reactions include swelling of the throat and airway, and difficulty in breathing. This

condition requires emergency medical treatment (Aberer et al., 2003).

In 2003 the value of milk discarded because of positive antibiotic test results

exceeded $7.6 million USD. Additionally, 8 of 54,932 antibiotic tests performed on

pasteurized fluid milk and milk products were positive resulting in disposal of 64,000

lbs (29,030 kg) of finished products (Boonk & Van Ketel, 1981; Ormerod, 1987).

There is also the possibility that antibiotics may be directly added to milk in an effort

to reduce the number of viable bacteria. That the problem is becoming more acute

seems evident from an F.D.A. survey completed early in 1955.This indicated that

11.6% of 474 samples of milk, collected nation-wide, contained measurable amounts

of penicillin. A survey made a year earlier found only 3.2% of 94 samples positive

(Goforth & Goforth, 2000).

11

2.4 The Genus Bacillus

Members of the Bacillus genus are generally found in soil and most of these bacteria

have the ability to disintegrate proteins, namely proteins with proteolytic activity.

Which contributes to the fertility of the soil. It was reported that members of the

species Bacillus generally produced polypeptide type bacteriocine antibiotics and

that these antibiotics generally affect gram (+) bacteria. It was also reported that

since most Bacillus species populate the same ecosystems as Streptomyces and other

antibiotic producers, they might have acquired resistance to antibiotics produced

under natural conditions (Aslim, SAĞLAM, & Beyatli, 2002).

2.4.1 Characters of bacillus species

Bacillus species are Gram-positive, endospore-forming, chemoheterotrophic rod-

shaped bacteria which are usually motile with peritrichous flagella; they are aerobic

or facultative anaerobic and catalase positive. Members of the Bacillus genus are

generally found in soil and represent a wide range of physiological abilities, allowing

the organism to grow in every environment and compete desirably with other

organisms within the environment due to its capability to form extremely resistant

spores and produce metabolites that have antagonistic effects on other

microorganisms (Amin, Rakhisi, & Ahmady, 2015).

The spores of thermophilic bacteria Geobacillus stearothermophilus (G.

stearothermophilus) are extremely resistant to high temperature and therefore, it is

the most frequently applied organism to select conditions for thermal sterilization of

food (Iciek et al., 2008).

12

Figure 2.1: Bacillus gram stained smear under light microscope (photograph by the

author)

2.4.2 Bacillus Species:

Bacillus species are divided in to three groups based on the morphology of the spore

and sporangium:

• Group 1 – Gram positive, produce central or terminal, ellipsoidal or cylindrical

spores that do not distend the sporangium: Bacillus anthracis, Bacillus cereus

(B.cereus), Bacillus mycoides, Bacillus thuringiensis and Bacillus megaterium.

• Group 2 – Gram variable with ellipsoidal spores and swollen sporangia: Bacillus

pumilus, B. subtilis, Bacillus circulans, Bacillus coagulans and Bacillus

licheniformis (B. licheniformis) Bacillus alvei, Bacillus brevis and Bacillus macerans

belonged to this group but have since been re-classified to other genera.

• Group 3 – Gram variable, sporangia swollen with terminal or subterminal spores:

Bacillus sphaericus (Rods, 2014).

2.4.3 B. cereus group and B. subtilis group

In recent years, there has been a taxonomic development in two selected groups of

the genus Bacillus. They are named the B. subtilis group and the B. cereus group.

The Bacillus genus commonly found in the environment. In line with this, although B

.licheniformis, B. pumilus, and B. mojavensis. Also, it has been classification of

Bacillus species to Groups Based on Phenotypic Similarities Group I. The B.

polymyxa group, Group lI. The B. subtilis group, Group IlI. The B. brevis group,

13

Group IV. The B. sphaericus group, Group V. The thermophiles, Group Vl.

Alicyclobacillus, Unassigned species (Cornelis, 2008; Rods, 2014).

The B. cereus group has six approved species B. anthracis, B. cereus, B. mycoides,

B. pseudomycoides, B. thuringiensis and B. weihenstephanensis (Bhandari, Ahmod,

Shah, & Gupta, 2013; Cornelis, 2008; Rods, 2014).

B. subtilis group includes B. subtilis subsp, B. subtilis subsp. spizizenii, B.

mojavensis, B. vallismortis, B. clausii, B. atrophaeus, B. amyloliquefaciens, B.

licheniformis, B. sonorensis, B. firmus, B. lentus and B. sporothermodurans

(Bhandari et al., 2013; Rods, 2014).

2.4.4 Bacillus used of production many enzymes

Thermophilic bacteria is one of the important sources that produce thermostable

enzyme, Bacillus strain is one of the main producers of protease and amylase of

potential industrial application. Proteases constitute one of the most important groups

of industrial enzymes, accounting for about 60% of the total enzyme market in the

world. Proteases are used in various industries such as pharmaceutical, detergent,

textile, food and sewage treatment (Mamo & Gessesse, 1999; Nascimento &

Martins, 2004; Patasik, Runtuboi, Gunaidi, & Ngili).

2.5 Detection methods of antibiotic residues in milk

Detection of antibiotic residues in milk and other food products of utmost

importance. Various means and techniques were employed to achieve this task. In

the following section we discussed the most common methods used to detect

antibiotic residues in milk and milk products.

2.5.1 Bioassay

2.5.1.1 Definition

Bioassay is defined as the estimation of the potency of an active principle in a unit

quantity of preparation or detection and measurement of the concentration of the

substance in a preparation using biological methods (i.e. observation of

pharmacological effects on living tissues, microorganisms or immune cells or

14

animal). Therefore, microbioassay is also regarded as bioassay. Recently,

biotechnology has also been considered for bioassay. Bioassay of products like

erythropoietin, hepatitis-B vaccine and many others is being done through

biotechnology (Goyal, 2008).

2.5.1.2 Principle of Bioassay

The basic principle of bioassay is to compare the test substance with the International

Standard preparation of the same substance and to find out how much test substance

is required to produce the same biological effect, as produced by the standard. The

standards are internationally accepted samples of drugs maintained and

recommended by the Expert Committee of the Biological Standardization of world

health organization (WHO). They represent the fixed units of activity (definite

weight of preparation) for drugs.

Biological variation problem must be minimized as far as possible. For that one

should keep uniform experimental conditions and assure the reproducibility of the

responses (Goyal, 2008).

2.5.1.3 Delvotest

The Delvotest is the best known microbial inhibitor test but it is less widely

recognized that several versions of this test exist. Delvotest is recognized as the gold

standard in antibiotic residue testing. The first version to be developed, in the 1970s,

was the Delvotest P, designed to detect ß-lactams.

A more recent development, the Delvotest SP, is capable of detecting a wider

spectrum of substances, notably sulphonamides, but also has increased sensitivity to

tylosin, erythromycin, neomycin, gentamicin, trimethoprim and other antimicrobials.

The Delvotest SP appears identical to the Delvotest P, the only difference being the

need to incubate the Delvotest SP for 2 ¾ hours (Montero et al., 2005).

Its known as 'microbial inhibitor' test, involve incubating a susceptible organism in

the presence of the milk sample. In the absence of an antibiotic, the organism grows

and can be detected visually by a color change resulting from acid production. In the

15

presence of an antibiotic, or any other inhibitor, the organism fails to grow and a lack

of a color change is observed (Montero et al., 2005).

Figure (2.2): Delvotest milk kit and incubator

2.5.1.4 Penzyme test

The Penzyme test is an enzyme assay. Carboxypeptidase causes a color change in the

content of the test vial in the absence of antibiotics and an orange/pink color appears.

With the presence of sufficient beta-lactam antibiotics in milk, the enzyme forms a

stable and inactive complex and the yellow color of the content of the vial remains.

The Penzyme test is simple and results are obtained in 20 min (Zeng et al., 1996).

Figure (21): Enzyme milk test

16

Figure (2.2): Results of Penzyme milk test

2.5.1.5 Copan test

The Copan test (CH ATK) P& S kit is a method for detection of antibiotic residues in

milk which has recently undergone a validation exercise in an independent Irish

laboratory. Copan test method is also based on the International Dairy Federation

IDF standard method for determination of antibiotic residues in milk. This method is

very similar to that of the Delvo®

SP test method, however, the nutrient tablet is

already added to the agar, therefore, the procedure is one step shorter than the

Delvo® SP (Ireland, 1999).

Figure (2.5): Copan SINGLE Test tubes and incubator

Negative Presumed Positive

17

Figure (2.3): Copan MICROPLATE

Positive result = Antibiotics are present

Negative result = No Antibiotics present

Figure (2.4): COPAN MILK TEST results

2.5.1.6 Charm Farm test

The Charm Farm test is a microbial inhibition test, which uses a one-step single

service vial. The Charm Farm test is a broad screening assay for five families of

veterinary drugs, including beta-lactams, sulphonamides, tetracyclines,

aminoglycosides and macrolides in raw, commingled, bovine milk.

The results are stable for 8 hours after assay completion and can be read by visual

color comparison or optionally with a pH meter. The Charm Auto-Farm Equipment

is required to run this test. The test can be completed in approximately 3.5 hours. Up

to 12 tests can be run simultaneously (Ireland, 1999).

18

Figure (2.8): Charm SL Beta-lactam Farm Test

2.5.1.7 Idexx parallux (antibiotic test kit)

The Parallux Antibiotic Test Kit is a capillary based solid-phase fluorescent

immunoassay for the determination of beta-lactams, sulphonamides and tetracyclines

in bovine milk. The system consists of an assay cartridge which contains four glass

capillaries, a reagent tray with four wells of dried reagents and a Parallux Processor

which processes the assay, reads fluorescence output, and reports test results. The

Parallux antibiotic residue test takes < 8 minutes to complete. Parallux identifies an

extensive range of antibiotics and sulphonamides, however, it is important to note

that the range of antibiotics and sulphonamides identified is dependent on the

specific assay cartridge used. Present chemical tests do not have sufficient

sensitivity to detect the minimum tolerable levels of antibiotic concentration, so there

are other more sensitive methods (Ireland, 1999).

19

Figure (2.5): Idexx parallux (antibiotic test kit)

2.5.1.8 Microbiological system in microtitre plates

multi-plate microbiological systems (MPMS) in Petri dishes that use different test

organisms (bacteria-tests) with adequate sensitivity to each antibiotic family. These

microbiological systems allow for the detection of an increased amount of molecules

and a subsequent classification in groups of antibiotics. The MPMS use three, four,

five, six, seven or eight Petri dishes of different compositions (Gaudin et al., 2004;

Nagel et al., 2013; Tumini, Nagel, Molina, & Althaus, 2017).

The sensitivity of this five-plate test, called Screening Test for Antibiotic Residues

(STAR), was established by the analysis of milk samples spiked with 66 antibiotics

at eight different concentrations (Gaudin et al., 2004).

2.5.2 ELISA

Enzyme linked immunosorbent assay (ELISA) technique is based on the antibody

sandwich principle. A rapid ELISA for the detection of penicillin G in milk at

concentrations of 6 ng/ml (0.01 IU/ml) was used to screen farm milk samples

previously reported as containing no detectable levels of antimicrobial substances. It

uses a single-well per test format and a percent inhibition cut-off for the

determination of positive/negative endpoint. Comparison with intra-assay standards

lowered the numbers of positives to 0.42%. Analysis of 170 milk samples positive

for antimicrobials confirmed 92.4% as containing penicillin G using the prescribed

cut-off level and 87.6% when compared to standards (Jackman et al., 1990).

20

2.5.3 Chromatography

While rapid screening tests are commonly used to detect the presence of antibiotics

in milk, more accurate chromatographic methods are required by government

regulatory agencies to identify and confirm the identity and quantity of antibiotic

present. Here it is possible to review recent developments in the chromatographic

determination of antibiotic residues in milk.

A simple and rapid ion-pairing liquid chromatographic method was developed for the

simultaneous determination of five penicillins (PCs), ampicillin (AB-PC),

benzylpenicillin (PC-G), cloxacillin (MCI-PC), dicloxacillin (MDI-PC) and nafcillin

(NF-PC) in milk. These PCs are most frequently used for the treatment of mastitis of

cows. These antibiotics were extracted with acetonitrile from milk and cleaned up by

solid-phase extraction with a C18 cartridge (Takeba, 1998).

An analytical method for the separation and detection of 12 aminoglycosides has

been optimized using two kinds of chromatographic conditions (HILIC and Ion

pairing). In Hydrophilic Interaction, ZIC_HILIC column was used, by which the

following parameters for the mobile phase were evaluated: concentration of

ammonium acetate buffer, percentage of formic acid and effect of acid type. The

comparison between the two separation methods shows that the response area of the

majority of analyses tested increases when using the ion pair mode. Also, the high

value of S/N and the lower detection limit (5 - 15 μg∙mL−1

) for most

aminoglycosides studied make the ion pairing method more preferable than HILIC

interaction (Mokh et al., 2014).

A simple and rapid reversed phase high performance liquid chromatograph (HPLC)

method for analysis of oxytetracycline (OTC) was developed and applied in the

determination of the antibiotic in fresh milk sample. Isocratic elution was performed

with acetic acid: water (pH 4.5): acetonitrile (4:68:28), using a polymer reversed-

phase (PLRP) column and UV detection at 354 nm wavelength. The method

demonstrated successful application for analysis of 100 milk samples. Two samples

out of 70 from livestock keepers tested OTC positive while none of the 30 samples

from milk centers tested positive (Kaale et al., 2008).

21

2.5.4 HPLC-MS/MS method

High pressure liquid chromatography coupled with UV detector (HPLC-UV) is the

technique usually adopted as a confirmatory method for antibiotic residues. This

technique has some limitations: mainly it has a low sensitivity and selectivity;

therefore, many purification steps are needed.

Martins-Júnior and others develop a simple and fast method to identify and

quantify fourteen antibiotics from different classes in milk, including five β- lactams,

four sulfonamides, three tetracyclines, one macrolide and one cephalosporin, using

reversed-phase liquid chromatography with electrospray ionization and triple

quadrupole mass spectrometry (MS/MS). Dicloxacillin and erythromycin showed the

lower and higher decision limits (cc) results of 0.05 and 9.77g L-1

, respectively.

Overall, the recoveries results ranged from 65 to 125%, with standard deviation

values from 2.0 to 15%. This method was also applied to evaluate the quality of

different fat milk brands offered in the Brazilian market (Martins-Júnior et al., 2007).

22

Chapter III

Materials and Methods

23

Chapter III

Materials and Methods

3.1 Materials

3.1.1 Apparatus

The apparatus used in this study are listed in table 3.1

Table (3.1): List of the apparatus used in this work

Apparatus Manufacture/model Country

Light microscope Leica China

Centrifuge 50 ml Hettich Germany

pH/ Meter Thermo Singapore

Autoclave Tuttnauer Germany

Incubator Memmert Germany

IR Concentrator N- Biotek Korea

Water bath

3.1.2 Chemicals, culture media and antibiotics

Seven types of media were used for carrying out this study are listed in table (3.2). In

addition, lactose 1%, glucose 1% and fructose 1% for supplement media, NaOH, and

HCl to adjust media pH. BromCresol Purple Indicator was used in the development

of Kit media. Table (3.3) contains a list of antimicrobials used for sensitivity test.

Table (3.2): Media used for isolation, cultivation, identification and kit

development

Media Manufacture Country

Brain Heart Infusion Broth HiMedia India

Muller Hinton Agar HiMedia

Nutrient Agar HiMedia

Blood agar HiMedia

Nutrient broth HiMedia

MR- VP HiMedia

Hicrome Bacillus agar Fluka analytical

24

Table (3.3): Antimicrobial discs which was be used in this study

Antimicrobial discs Potency Antimicrobial discs Potency

Amoxicillin 15 mcg Co-Trimoxazole 25 mcg

Amoxyclav 30 mcg Erythromycin 15 mcg

Azithromycin 15 mcg Gentamicin 10 mcg

Cefadroxil 30 mcg Neomyicin 30 µg

Cefalexin 30 mcg Norfloxacin 10 mcg

Cefazolin 30 mcg Novobiocin 30 µg

Cefixime 5 mcg Oxacillin 1 mcg

Cefotaxime 30 µg Penicillin G 10 mcg

Chloramphenicol 30 mcg Tetracycline 30 mcg

Cloxacillin 1 mcg

3.1.3 Microorganism maintenance and storage

Bacillus from the commercial kit was used in some of the trials. Organism (116) that

was isolated from collected samples from different areas, and was maintained in

Brain Heart Infusion Agar medium slant at 2-8 0C.

3.2 Methodology

3.2.1 Soil Sample collection

In order to isolate candidate bacteria that are antibiotic sensitive and capable of

growing at high temperature, soil samples were collected from different regions of

Gaza strip (table 3.4). The samples (approximately 20 g each) were collected using

sterile cups. All samples were transferred to the microbiology research laboratory

under sterile conditions (Amin, Rakhisi, & Ahmady, 2015).

25

Table (3.4): Locations for the collected soil samples

N City Longitude Latitude N City Longitude Latitude

1 Gaza 31,513198 34,4400208 16 Rafah 31,267830 34,273273

2 Rafah 31,270537 34,265919 17 Rafah 31,267830 34,273273

3 Gaza 31,512394 34,448226 18 Rafah 31,267723 34,273290

4 Gaza 31,513340 34,449733 19 Rafah 31,267724 34,273291

5 Gaza 31,516590 34,437790 20 Rafah 31,267735 34,273300

6 Gaza 31,513306 34,441746 21 Rafah 31,267741 34,273305

7 Rafah 31,269512 34,267125 22 Rafah 31,267742 34,273306

8 Rafah 31,269664 34,268526 23 Rafah 31,267743 34,273307

9 Rafah 31,269788 34,270973 24 Rafah 31,267744 34,273308

10 Rafah 31,269920 34,271641 25 Rafah 31,267740 34,273309

11 Rafah 31,267667 34,272319 26 KhanYounes 31,298821 34,355325





3.2.2 Isolation of Bacillus spp.

One gram of each soil samples was added to 5 mL of nutrient broth, mixed by

vortexing and heated at 80oC for 10 minutes. After that, tubes were cooled rapidly

under tap water. After the cooling, 0.1 mL of the supernatant of each tube containing

suspension of soil and culture media was inoculated on nutrient agar plates by

streaking. Plates were incubated at 37oC for 24 hours. From each plate, one or more

of well separated colonies were picked and subcultured onto the surface of Blood

agar plates to ensure purity (Amin et al., 2015).

3.2.3 Antibiotic susceptibility by agar diffusion method

Each of the isolates was standardized using colony suspension method. Each

strain’s suspension was matched with 0.5 McFarland standards to give a resultant

concentration of about 1.5 × 108 cfu/mL. The antibiotic susceptibility testing was

determined using the modified Kirby–Bauer diffusion technique, by swabbing the

26

Mueller-Hinton agar (MHA) plates with the resultant Brain Heart Infusion Broth

suspension of each strain, antibiotics alone and their combinations taking care not

to allow spillage of the solutions on to the surface of the agar. The plates were

allowed to stand for at least 30 min before being incubated at 37°C for 24 h. The

determinations were done in duplicate. After 24 h of incubation, the plates were

examined for zones of inhibition. The diameter of the zones of inhibition produced

by the antibiotic and their combinations was measured and interpreted using the

CLSI zone diameter interpretative standards. Antimicrobial discs which were used

for this study are listed in table (3.1) (Amin et al., 2015; Aslim et al., 2002; Yilmaz,

Soran, & Beyatli, 2006). One hundred nineteen isolates were tested. Only those that

showed high susceptibility to antimicrobials (34 isolation) were selected for further

testing.

3.2.4 Thermal resistance studies and identification

3.2.4.1 Growth at 60 degrees Celsius

Candidate isolates were streaked on Nutrient Blood agar and were inoculated into

NB and BHIB tubes and incubated at 55-60°C for 24 h. Because of the high

temperature of incubation, plates were placed in a plastic bags to avoid evaporation

of the sample and to preserve the shape of the colonies. Only one isolate (Isolate no.

25.5) was selected because it exhibited good growth on both liquid and solid media.

3.2.4.2 Bacterial culture and identification

For bacterial cultural characterization, a one colony of isolate 25.5 was transferred in

to pre-labeled blood agar. The inoculated plates was incubated at 60oC for 24 hours,

after which their cultural characteristics were observed and recorded. The isolates

then identified by colony morphology and characteristic growth, gram stain, spore

stain, Beta hemolysis on blood agar, triple sugar iron agar reaction and pattern of

biochemical profile (Catalase, Oxidase, Starch hydrolysis, Methyl Red, Voges-

Proskauer (MR-VP), Sulfide Indole Motility, and Lysine Iron Agar, Urease, and

Citrate test) in accordance with the standard methods.

27

3.2.5 Test Media formulation

For the purpose of formulation of an appropriate media for the detection of Bacillus

growth in the absence of antimicrobials, several media composition were attempted

(Table 3.5). The growth-initiating potential of experimental media was assessed by

comparison with that obtained with the control Blood agar and Nutrient agar

medium. Other media were prepared by adding sugar. Temperature and pH effects

experiments were also performed

Each of the liquid formulations was tested for their ability to support and detect the

growth of the selected isolate. Liquid media (2 ml) was placed in screw capped

tubes. Negative control (milk tested negative with commercial kit) and positive

control (Antimicrobial was deliberately added) were used to inoculate 2 different

tubes of each formulation and one tubes of the commercial kit for comparison. All

tubes were incubated at 60oC for 24 hours. Results were observed and recorded as

color change from purple to yellow (Purple -no growth) is positive for antimicrobial

residues while yellow color development (growth) is considered negative for

antimicrobial residues.

Table (3.5): Various media formulation which was used in this study

N Name of media

pH

Tem

peratu

re

Added

sugar

Bro

mocry

sol

purp

le

Quan

tity/tu

be

1 Nutrient Broth 6.7 65oC x √ 5 ml

2 Brain Heart Infusion Broth 6.7 65oC x √ 5 ml

3 Nutrient Broth 6.5 60oC 1% glucose √ 5ml

4 Nutrient Broth 6.5 60oC 1% fructose √ 5 ml

5 Nutrient Broth 6 60oC 1% lactose √ 5 ml

6 Nutrient Broth 6 60oC 1% lactose √ 2 ml

7 MR-VP 6 60oC 1% lactose √ 2 ml

8 MR-VP 6 60oC x √ 2ml

MR-VP= Methyl Red-Voges Proskauer medium

28

3.2.6 Determination of antimicrobial residues and microbial load in milk

samples

3.2.6.1 Milk samples

A total of 81 milk samples were collected randomly from different farms in Gaza

strip. One hundred ml were collected from each cow in sterile cup, labeled and then

placed in an ice box and transferred to the Islamic University Microbiology research

laboratory within one hour from collection.

3.2.6.2 Preparation milk sample

Each milk sample was examined for their total bacterial count (section 3.2.6.3). The

remaining of the milk sample was divided into four parts. The first part was screened

for the presence of antimicrobial residues using a commercial kit (section 3.2.6.4).

While, the second part was screened by the locally developed kit (section 3.2.6.5).

The third part was screened by the locally prepared testing broth media and kit

bacteria (Hybrid kit) (section 3.2.6.6). The fourth part was screened by the locally

prepared testing agar media in plats. Both the commercial kit bacteria and the

candidate bacteria were used (section 3.2.6.7).

Table (3.6): Distribution of milk samples by governorates

Governorate Number of samples Number of farms

Rafah 13 samples 2 farms

KhanYounes 18 samples 2 farms

Middle 5 samples One farm

Gaza 45 samples 4 farms

3.2.6.3 Total bacterial count

One ml of well-mixed milk sample was added to 9 ml of sterile buffered saline

diluents to make 10-1

dilution. Serial dilution was then followed to make 10-2

to 10-6

dilutions. 0.1 ml from each dilution was plated onto the surface of Nutrient Agar

plate. L-shaped glass rod was used to spread the liquid over the entire surface of the

agar. Plates were incubated at 37oC for 48 hours. Plates containing counts between

30-300 were selected and counted. The total bacterial count was then calculated

using the following formula.

Total plate count = Number of colonies X 1/dilution X 1/0.1

29

3.2.6.4 Antimicrobial residues using commercial kit

Rapid antibiotic test (MiRA Test-Ref. 80355) is a cultural test that employs

Geobacillus stearthermophillus spores, microorganism with sensitivity at broad

spectrum towards a large number of antimicrobials, and can be used as screening

method to search of residual antibiotics, in foodstuffs matrix like milk, meat, fish and

others.

Testing procedures was performed according to the manufacturer's instructions. Each

batch of rapid antibiotic test is subjected to quality control using milk specimen

containing antibiotics like gentamicin, penicillin G and tetracycline.

Test procedure

1- Remove the cup from the tube and Add 1 disc of spores to the medium.

2- Pre-incubate for 20min at 64 ± 0.5 °C in water bath or termoblock.

3- Remove the vial from the incubator and let it reach room temperature.

4- Introduce 1 mL of the milk sample.

5- Reintroduce the vial in the water bath or in the Termoblock at 64 ± 0.5 °C for

the second incubation for 4h – 6h.

3.2.6.5 Antimicrobial residues using locally developed kit

Locally developed consisted of vials containing 2 ml of indicators media in a screw-

capped vials. Kit bacteria was impregnated in filter paper disks and dried. For the

performance of the test, one ml of milk was transferred to the vial, and one filter

paper disk containing the bacteria was added. Vials was incubated at 60°C. The

results were recorded after 4 and 24 hours.

3.2.6.6 Antimicrobial residues using Hybrid kit

Hybrid kit consisted of screw capped vials containing 2 ml of the locally developed

kit media and the commercial bacterial disks obtained from the commercial kit. The

same procedures were followed as in section 3.2.6.6.

30

3.2.6.7 Antimicrobial residues using solid media

A well diffusion assay method for testing antibiotics in milk was developed wherein

Bacillus was used as the test organism and modified MR-VP (mMR-VP) agar media

was used as the test media. This was carried out using the previous procedure (Agar

well diffusion method). The method measures microbial growth inhibition by

antibiotic of Bacillus. modified MR-VP (mMR-VP) agar media plated were swabbed

with a standardized lawn of the test organism. Holes where punched in the agar using

sterile pasture pipette. 100 µL of the each of the test milk was added to a specified

hole. The plates were allowed to settle for 30 minutes at the refrigerator to allow for

extract diffusion and then incubated at 60oC for 24 hours. The zone of inhibition

around each milk sample was measured and recorded. Plates were read and zone was

determined by recording the distance of the well that preceded the yellow color

appearance (The development of yellow color indicate bacterial growth).

3.2.7 Statistical analysis

Statistical analysis was performed with PASW SPSS Statistics for Windows (version

18.0, NY, USA). The results were summarized and reported as percentages,

frequencies, means and standard deviations. The results were also presented through

charts and tables. The statistical analysis were performed by using Kruskal Wallis

ANOVA for multi-group comparisons, Mann-Whitney U-test for independent

samples, chi-square test of homogeneity for multi-group comparisons, chi-square

goodness-of-fit test and by Wilcoxon Signed Ranks test for paired comparisons. The

levels of agreement between the tests and its associated P-value were calculated

using by the Cohen’s Kappa statistic of agreement and evaluated using Landis-Koch

scale (Landis & Koch, 1977)P values less than 0.05 were considered significant.

31

Chapter IV

Results and Discussion

32

Chapter IV

Results and Discussion

In the present study, a new and improved (simple, inexpensive and easy-to-use)

microbial growth inhibition tests have been developed for the determination of the

presence or absence of antibiotic residues in milk samples.

4.1 Isolation of test microorganism

Table 4.1 shows a summary of the number of isolated strains from the soil samples

collected from the three governorates at Gaza strip. A total of 116 bacterial strains

were isolated from the 29 soil samples collected from various regions in Gaza strip

and cultured on an appropriate culture medium. The most isolated strains were from

Rafah (62 isolates), followed by Khan Younis (37 isolates) and then Gaza city (17

isolates).

Table (4.1): Number of soil samples and isolates obtained from the three

governorates at Gaza strip

Governorate No. of soil samples No. of isolates

Rafah 19 62

Khan Younis 5 37

Gaza 5 17

Total 29 116

4.2 Determination of the most antibiotic-sensitive strain

The susceptibility of all isolates (n = 116) against 19 of commonly used clinical

antibiotics were evaluated by disc diffusion method on Mueller Hinton agar plates to

determine their sensitivity (Figure 4.1). Among the isolated strains, 25 strains were

sensitive to eighteen antibiotics and nine were totally sensitive to all nineteen tested

antibiotics. The antibiotics used were belong to 19 antibiotic with different modes of

actions, including Beta-Lactams, (Penicillin G, Amoxicillin, Amoxyclav, Cloxacillin,

Oxacillin). Cephalosporin family (Cefalexin, Cefadroixl, Cefotaxime, Cefazolin,

Cefixime), Aminocoumarin (Novobiocin), Macrolides (Azithromycin and

Erythromycin), Chloramphenicols (Chloramphenicol), Sulphaamides (Co-

33

Trimoxazole), Fluoroquinolone (Norfloxacin), Tetracyclines (Tetracycline) and

Aminoglycosides (Neomyicin and Gentamicin ) (For more details see Appendix 1).

Figure (4.1): Antibiotic sensitivity patterns among the isolated strains

4.3 Determination of the best temperature-resistant isolate

The 34 strains recognized as highly sensitive to the tested antibiotics were selected to

test their temperature resistance properties at 60oC using various agar and broth

media (Table 4.2). Only one isolate (no. 25.5) exhibited good growth on all media

tested (both liquid and solid media). Other isolates, such as 7.1 and 12.3 showed

growth in NB medium only.

0

5

10

15

20

25

30N

um

ber

of

sen

seti

ve i

sola

tes

Number of antibiotic tested

34

Table(4.2): Growth evaluation of the 34 isolates on different media at 60oC

Nu

mb

er o

f

isola

tion

bact

eria

Nu

trie

nt

blo

od

ager

(N

BA

)

Nu

trie

nt

Bro

th

(NB

)

Bra

in h

eart

infu

sion

B

roth

(BH

IB)

Nu

mb

er o

f

isola

tion

bact

eria

Nu

trie

nt

blo

od

ager

(N

BA

)

Nu

trie

nt

Bro

th

(NB

)

Bra

in h

eart

infu

sion

B

roth

(BH

IB)

1.1 × × × 8.1 × × ×

2.2 × × × 8.2 × × ×

2.4 × × × 8.3 × × ×

3.2 × × × 11.1 × × ×

4.2 × × × 11.2 × × ×

5.2 × × × 12.3 × √ ×

6.1 × × × 12.4 × × ×

7.1 × √ × 13.5 × × ×

7.2 × × × 14.1 × × ×

7.3 × × × 16.2 × × ×

7.4 × × × 16.3 × × ×

7.5 × × × 16.7 × × ×

21.3 × × × 27.2 × × ×

21.6 × × × 27.9 × × ×

24.3 × × × 29.4 × × ×

25.5 √ √ √ 29.7 × × ×

26.9 × × × 29.9 × × ×

On the basis of these results the strain named 25.5 was chosen for further

characterization to assess their potential use in developing microbiological inhibition

tests for the determination of the presence or absence of antibiotic residues in milk

samples.

4.4 Characterization of isolate 25.5

The strain designated 25.5 was characterized based on morphological, physiological

properties according to Bergey’s Manual of Systematic Bacteriology. Data shown in

35

Table 4.3 indicate that, the isolate was Gram +ve, motile, and rod-shaped bacilli. It

was also found to be a spore former with subterminal, round to oval in shape spores.

The isolated bacterium tested positive for methyl red where it developed a red color

within 2 minutes and for starch hydrolysis test. The results were negative for Indole

test, Voges–Proskauer tests, H2S production, gas and acid from glactose, lactose,

sucrose and Urea hydrolysis test. It was also negative for ammonium dihydrogen

phosphate and sodium citrate utilization as their sole sources of nitrogen and carbon

respectively. The cultural, cellular, physiological and biochemical characteristics

exhibited by this isolate suggests that it belongs to B. subtilis.

Table (4.3): Phenotypic characteristics of the selected isolate (25.5)

Properties/test Results

Morphological characteristics

Spore morphology Sub-terminal, round to oval

Motility +

Gram stain +

physiological characteristics

Methyl red +

Voges-Proskauer test -

Indole production -

H2S production -

Starch hydrolysis +

Nitrogen source utilization

Ammoniam dihydrogen

phosphate

-

Urea hydrolysis -

Carbon source utilization

Sodium citrate -

Galactose +

Lactose -

+ Indicates positive and - negative.

36

Reviews of Bacillus distribution in the various environments suggest that the species

B. subtilis is widely distributed and ubiquitous organism throughout the different

environments, particularly in soil and air. For example, Amin, Rakhisi and Ahmady

(2015) reported that in 50 soil samples tested, only 30 isolates of Bacillus spp. were

obtained. These bacteria were classified into four spices including B. cereus (86.6%),

B. subtilis (6.6%), B. thuringiensis (3.3%) and B. pumilus (3.3%) (Amin et al., 2015).

In another study, forty bacteria were isolated from soil samples from six different

regions. Strains were identified as Bacillus species; namely, B. subtilis, B.

megaterium, B. firmus , B. sphaericus , B. thuringiensis, B. pumilus and Bacillus spp.

(Aslim et al., 2002).

In another study, carried out to screen amylase producing microorganism from soil

(Singh, Sharma, & Sharma, 2015). Bacillus megaterium from soil of different Agro

climatic zones(Reddy, Mohan, Nataraja, Krishnappa, & Abhilash, 2010). It has been

reported that isolating soil bacteria capable of blocking quorum sensing by

inactivating N-acylhomoserine lactones, bacteria degrading N-acylhomoserine

lactones were isolated from a Malaysian soil sample(Chan, Tuew, & Ng, 2007).

In later study, Bacillus sp. isolated from local marine samples collected from Saudi

Arabia for bacteria producing protease enzyme (Alnahdi, 2012).In another study,

four novel bacterial strains of what genus were isolated from cryogenic tubes used to

collect air samples (Shivaji et al., 2006).

A thermophilic microorganism; Bacillus thermoleovorans ID-1, isolated from hot

springs in Indonesia (Lee et al., 1999). The wide range distribution of this genus in

various environments is due to its capacity to grow over a wide range of conditions

including temperatures and pH . For example, Iciek, Blaszyk and Papiewska (2008)

reported that the survival of spores (Bacillus stearothermophilus) was investigated in

media (tryptone solution, red beet juice) of natural pH or acidified with organic acid

at pH ranging from 6.0 to 4.0. Thermal sterilisation was carried out in the

temperature range from 115°C to 125°C (Iciek et al., 2008).

37

In another study, B. subtilis JS-2004 isolated strain to produce α-amylase Studies on

crude a-amylase characterization revealed that optimum activity was at pH 8.0 and

70ºC (Asgher, Asad, Rahman, & Legge, 2007). In another study, that spore-forming

bacteria was isolated from a soil sample, Growth occurred at pH values ranging from

6.5 to 9.0, and optimum growth occurred at about pH 7.0. The optimum temperature

for growth was around 55ºC, and the upper temperature limit for growth was around

70ºC (Souza & Martins, 2001).

Strains belong to this genus are also able to form resistant-endospore and produce

antimicrobial compounds inhibitory to other competing and pathogenic

microorganisms. Arguelles-Arias, et all(2009) The genome of the plant-associated B.

amyloliquefaciens GA1 was sequenced. Several gene clusters involved in the

synthesis of biocontrol agents were detected (Arguelles-Arias et al., 2009). In

another study, In the context of biocontrol of plant diseases, the three families of

isolated found prodcing Bacillus lipopeptides – surfactins, iturins and fengycins were

at first mostly studied for their antagonistic activity for a wide range of potential

phytopathogens, including bacteria, fungi and oomycetes (Ongena & Jacques, 2008).

4.5 Antibiotic susceptibility test of the Bacillus subtilis isolate

Table 4.4 shows susceptibility test of the selected isolate B. subtilis against the 19

antibiotics. Based on the diameter of the inhibition zone, the organism exhibited

highly susceptibility to Oxacillin (44 mm), Chloramphenicol (35 mm),

tetracycline(30mm), gentamicin and penicillin G (25 mm), amoxyclav and

amoxicillin (24 mm), and cefotaxime (18 mm).

38

Table (4.4): The antibiotic sensitivity of B. subtilis isolate

An

tibio

tic

Pen

icillin G

Am

ox

icillin

Am

ox

ycla

v

Clo

xa

cillin

Ox

acillin

Cefa

lexin

Cefa

dro

ixl

Cefo

tax

ime

Cefa

zolin

Cefix

ime

No

vo

bio

cin

Azith

rom

ycin

Ery

thro

my

cin

Ch

lora

mp

hen

icol

Co

-Trim

ox

azo

le

No

rflox

acin

Tetra

cyclin

e

Neo

my

icin

Gen

tam

icin

Sy

mb

ol

P

AM

L

AM

C

CX

OX

CL

CF

R

CT

X

CZ

CF

M

NO

AZ

M

E

C

CO

T

NO

R

TE

N

GE

N

Disc

po

tency

(mg

)

10

15

30

1

1

30

30

30

30

5

30

15

15

30

25

10

30

30

10

zon

e of

inh

ibitio

n (m

m)

25

24

24

R

44

32

30

18

22

9

22

30

20

35

35

30

30

26

25

R: No zone

The observed susceptibility to antibiotics is in agreement with several studies who

found that strains of B. subtilis exhibited high susceptibility to wide range of

antibiotics (Barbosa, Serra, La Ragione, Woodward, & Henriques, 2005) and only

few of the reported strains were described as antibiotic-resistant strains (Bernhard,

Schrempf, & Goebel, 1978).

The results of Aslim et. al (2002) for example showed high susceptibility rate of

Bacillus strains isolated from different soil samples to vancomycin, chloramphenicol,

tetracycline, gentamicin erythromycin, cephalothin and ampicillin, and were (100%),

(97%), (93%), (90%), (80%), (40%) and (23%) respectively. Similar findings were

reported in another study; where the resistant rates to kanamycin (79%), vancomycin

(65%), tetracycline (26%), penicillin G (23%), erythromycin (16%) and

chloramphenicol (11%) (Temmerman, Pot, Huys, & Swings, 2003).

Generally, microbes which exhibit detectable growth inhibition in the presence of

antibiotics may be useful to be used in microbial inhibition tests. In addition to B.

subtilis, the most widely used microbes for such an application include:; B.

megaterium; S. aureus; P. aeuginosa; E. coli; and B. licheniformis (Aslim et al.,

2002).

39

4.6 Media formulation

Evaluation of the optimum cultural and test conditions that enable the multiplication

of the selected test microorganism in the absence of antibacterial compounds was

made by using different cultural conditions including three kinds of broth media

(NB, BHIB, MR-VP) supplemented with different carbohydrate sources. The

bacterial growth was assessed at two different temperatures and three pH levels

(Table 3.5). Bromocresol purple was added to the media to allow the detection of

growth of test microorganism via a color change from purple to yellow when

organisms grow and ferment the carbohydrates source contained in the medium

lowering the pH of the medium to acid.

B. subtilis grew equally well in both Methyl Red-Voges Proskauer media (Medium 7

and 8) adjusted at pH 6 and incubated at 60°C. Lactose was omitted from the later

medium formulation because its absence does not affect the bacterial growth and

because a considerable amount of this sugar is already found in milk, where it

constitutes about 2–8% of milk (by weight). Hereinafter, the test medium will be

named as modified MR-VP (mMR-VP) agar media. Other media formulations such

as No. 1, 2, 3, 4, 5 and 6 did not support any bacterial growth under the tested

circumstances.

The results of the present study suggest that B. subtilis is very suitable as test

organism because it is very sensitive to large number of antibiotics such as

Oxacillin, Chloramphenicol and Co-Trimoxazole. Additionally, this species have the

advantage that it grow well at high temperature (60°C) and this is in contrary to

many microorganisms which cannot grow at this temperature and therefore there is

little possibility that other organisms which are possibly present in the milk would

affect the result of the test. As Bacillus species, the test organism may be adsorbed to

paper disc and added to liquid test medium as a separate source. Moreover, when

tests is applied with an agar medium, as Bacillus strain, it could be easily

incorporated into the agar medium prior to solidification, making it possible to

perform the test easily in solid media.

40

4.7 Microbial quality of milk

A total of 81 milk samples were collected from four governorates at Gaza strip as

shown in Figure 4.2

Figure (4.2): The number of milk samples collected from the four governorates at

Gaza strip

4.7.1 Microbial quality of milk based on TBC

The total bacterial counts (TBC) results in milk samples were used to calculate the

arithmetical means, geometric means and standard deviations as well as minimal and

maximal values of mesophilic bacteria. The basic statistical measurements were

determined in the Table 4.5. TBC in all examined milk samples ranged from 0.00 to

3.0 × 1010

ml-1

with the arithmetical mean of 1.1 × 109 ml

-1. Statistically, Kruskal-

Wallis test proved a statistically highly significant difference in the total bacterial

count across the governorates (p < 0.001). The lowest average of TBC values were

found in samples of milk collected from Rafah (7.3×104) compared to Khan Younis,

Middle governorate and Gaza governorates, where the average total bacterial count

were 3.0×105, 2.5×10

5 and 2.0×10

9 ml

-1 respectively.

13

18

5

45

Rafah Khan Younis Middle Gaza

41

Table (4.5): The basic statistical measurements for the number of micro-

organisms per milliliter of 81 tested samples

Governorate No. of

samples

Arithmetic

mean

Geometric

mean

Range

Maximum-

Minimum

Rafah 13 7.3×104 5.7×10

2 0.0 – 8.0×10

5

Khan Younis 18 3.0×105 2.0×10

4 1.0 - 2.5×10

6

Middle 5 2.5×105 2.0×10

5

8.4×104

-

4.3×105

Gaza 45 2.0×109 3.9×10

6 2.0 - 3.0×10

10

Total 81 1.1×109 2.4×10

5 0.0 - 3.0×10

10

Except for Gaza and Middle governorates (p > 0.05), the multiple comparison

(Mann-Whitney U and Wilcoxon W test) of governorates shows a statistically highly

significant difference in TBC between all other governorates, between Rafah and

Middle governorate (p = 0.007), and a statistically significant difference (p = 0.031)

between Rafah and Khan Younis, between Rafah and Gaza (< 0.001), between

Middle governorate and Khan Younis (p = 0.044) and between Khan Younis and

Gaza (< 0.001) (Table.4.6).

Table (4.6) : Statistical differences in TBC in relation to the governorates (Based

on Mann-Whitney U/Wilcoxon W test)

Middle governorate Khan Younis Gaza

Rafah 0.007 0.031 < 0.001

Middle governorate 0.044 0.141

Khan Younis < 0.001

The TBC in milk samples was used to assess the microbial quality of milk in the

Gaza strip on the basis of the standards prescribed by the Palestinian Dairy Products

Regulations. According to the Regulations, the total number of microorganisms

should not exceed 105 colony forming unit per ml of raw cow’s milk. The results

were categorized as “passed” or "failed". The sample was considered failed when

42

the count of aerobic bacteria present in excess of 105 cfu/mL, otherwise, it will pass

the legal standards. Out of the 81 milk samples examined in the study, 41 (50.6%)

failed (i.e. exceeded 105 cfu/mL) the legal standards, while 40 (49.4%) passed the

TBC standards for milk (Table 4.7). In comparison between the four governorates,

7.7% (1/13), 16.7% (3/18), 60.0% (3/5) and 75.6% (34/56) of milk samples

collected from Rafah, Khan Younis, Middle and Gaza governorates respectively,

were present in excess of 105 cfu/mL i.e. they failed to comply with the legal

standards prescribed by the Palestinian Dairy Products Regulations for TBC (Table

4.7). These results indicate that the bacterial abundance and consequently the

microbial quality was affected by the region from which the milk samples collected.

Table (4.7): Microbiological quality of fresh cattle milk from different

governorates of Gaza strip as judged by legal standards of Palestinian Dairy

Products Regulations

Governorate Passed Failed* Total

Rafah 12 (92.3%) 1 (7.7%) 13

Khan Younis 15 (83.3%) 3 (16.7%) 18

Middle governorate 2 (40.0%) 3 (60.0%) 5

Gaza 11 (24,4%) 34 (75.6%) 56

Total 40 (49.4%) 41 (50.6%) 81

* Aerobic plate count > 105 cfu/mL

The higher values of microbial contamination of milk from certain areas may be

connected with poor cows cleaning and improper milking that were consequently

reflected in TBC values.

Bacterial contamination of raw milk can generally occur from three main sources;

inside the udder (infections e.g., mastitis), outside the udder (skin contaminants), and

from the surface of equipment used for milk storage and handling. Cow health,

environment, milking procedures and equipment sanitation can influence the level of

microbial contamination of raw milk. Equally important is the milk temperature and

length of time milk is stored before testing and processing that allow bacterial

contaminants to multiply. All these factors will influence the total bacteria count

(TBC) and the types of bacteria present in raw milk.

43

4.8 Milk testing for antibiotic residues, test performance characteristics and

residue prevalence

The objectives of these experiments were to assess and compare the performance of

the locally developed test on fresh milk samples by running the test in parallel with

the standard MiRA Test currently used for the routine testing of residue in fresh milk

samples and to determine the proportions of positive and negative antibiotic residues

samples, as well as to find out and assess the level of agreement between the newly

developed tests and MiRA Test by using the Kappa statistic of agreement.

Figures 4.3 and 4.4 show the percentage of presumably positive and negative results

recorded for the six experimental trials after 4 and 24 h experimental periods. The

percentages of presumably positive results after 4 hours experiment were 82.7, 79.0

and 76.5 for the LDMBB-4 h, LDMBG-4 h and MiRA Test-4 h respectively (Figures

4.3). After 24 h, this percentages dropped to 49.4, 35.8 and 17.3% in LDMBB-24 h,

LDMBG-24 h, and MiRA Test-24 h respectively (Figures 4.4).

Figure (4.3): Overall proportion of presumably of positive and negative results

(%) across the experimental groups after 4 h test period

44

Figure (4.4): Overall proportion of presumably of positive and negative results

(%) across the experimental groups after 24 h test period

Results of the chi-square test of homogeneity revealed a statistically significant

difference (p < 0.001) in proportions of positive (or negative) residues in milk

samples between the six experimental trials; the locally developed medium broth

with B. subtilis (LDMBB-4 h), the locally developed medium broth with G.

stearthermophillus LDMBG-4 h, the MiRA Test after 4 h test interval (MiRA Test-4

h), the locally developed medium broth with B. subtilis (LDMBB-24 h), the locally

developed medium broth with G. stearthermophillus (LDMBG-24 h) and the MiRA

Test after 24 h test interval (MiRA Test-24 h). Table 4.6 shows the distribution of

presumably positive and negative results for the six experimental trials.

Table (4.8): Results (expressed as number of positive and negative) of analysis of

milk samples by using the six experimental trials

Experiment

Frequency of positive

and negative results

Negative Positive

4 h test period

Locally developed medium broth with B. subtilis

(LDMBB-4 h)

14 67

Locally developed medium broth with G.

stearthermophillus (LDMBG-4 h)

17 64

MiRA Test-4 h 19 62

24 h test period

45

Locally developed medium broth with B. subtilis

(LDMBB-24 h)

41 40

Locally developed medium broth with G.

stearthermophillus (LDMBG-24 h)

52 29

MiRA Test-24 h 67 14

When antibiotics residues were determined using the MIRA test (Fig. 4.5), 62 of the

81 milk samples tested positive for presence of antibiotics after 4 hour interval (the

period recommended by the manufacturer).

Positive Negative

Figure (4.5): MIRA test results after 4 h incubation period (purple color

indicates positive result and yellow color indicates negative result)

No difference was found in the distribution of positive and negative samples between

the MiRA Test-4 h and those obtained by LDMBB-4 h (P = 0.190) and LDMBG-4 h

(P = 0.60, chi square goodness of fit test) tests.

After 24 h test period, the numbers of presumably positive milk samples obtained by

MiRA Test-24 h were reduced to 14, while those of negative samples was increased

to 67. This is statistically significant different (P < 0.001) from the results obtained

by the locally developed test (LDMBB-24 h) in which the distribution of number of

positive to negative milk samples were 40 and 41 respectively (Table 4.8). This may

46

indicates that the locally developed test is more stable than the commercial MIRA

test.

The comparison of MiRA Test-4 h and LDMBG-4 h tests showed that both tests are

positive in 75.3% of the milk samples, while 19.8% of the samples were negative in

both tests. For MiRA Test-4 h and LDMBB-4 h tests, 75.3% of the samples were

detected positive in both while 16.0% were negative in both tests (Table 4.9).

47

Table (4.9): Numbers and percentages (%) of positive (+) and negative (-)

results of commercial test (MiRA Test-4 h) and locally developed medium broth

with G. stearthermophillus (LDMBG-4 h), and commercial test and locally

developed medium broth with B. subtilis (LDMBB-4 h)

Developed test MiRA Test-4 h

- +

LDMBG-4 h

- 16

19.8%

1

1.2%

+ 3

3.7%

61

75.3%

LDMBB-4 h

- 13

16.0%

1

1.2%

+ 6

7.4%

61

75.3%

Despite their importance in interpretation the results, when a newly test is developed

however, it is necessary to compare its results to those from standard test by using

statistical analysis other than those mentioned above. Comparison of a new test

technique with a newly established one is often needed to check whether they agree

sufficiently for the new to replace the old. Cohen’s kappa statistic (κ) is commonly

used in such situation where it is used to assess the agreement between alternative

methods of categorical assessment (such as positive/negative) when new techniques

are under study (Martin, Meek, & Willeberg, 1987).

The kappa statistic of agreement measures the agreement beyond that expected due

to chance. Accordingly, this statistic was used to recognize the level of agreement

between the commercial MiRA Test and the locally developed test in addition to

other experimental trials (Table 4.10). The levels of agreement were assessed at two

times durations (4 and 24 h) and results interpretation was made according to the

Landis-Koch scale (Landis and Koch, 1977). A kappa of 0, 0.01-0.20, 0.21-0.40,

0.41-0.60, 0.61-0.80 and 0.81-1.00 indicates poor, slight, fair, moderate, substantial

and almost perfect level of agreement respectively.

48

After 4 hours test period, the highest obtained level of agreement (k = 0.857) was

between MiRA Test-4 h and LDMBG-4 h, suggesting almost perfect agreement

between the two tests. At the same test period, there was a substantial level of

agreement (k= 0.735) between the locally developed test (LDMBB-4 h) and the

commercial test (MiRA Test-4 h).

Table (4.10): Kappa statistics of agreement between MIRA Test and other

locally developed and modified tests and its interpretation according to the

Landis Koch scale

Test

duration

(h)

Test pair Kappa

Evaluation of

Kappa according to

the Landis-Koch

scale

P-value

4

MiRA Test-4 h and

LDMBB-4 h

0.735 Substantial

agreement

< 0.001

MiRA Test-4 h and

LDMBG-4 h

0.857 Almost perfect

agreement

< 0.001

4 vs. 24

MiRA Test-4 h and

MiRA Test-24 h

0.120 Slight agreement 0.023

MiRA Test-4 h and

LDMBB-24 h

0.362 Fair agreement < 0.001

MiRA Test-4 h and

LDMBG-24 h


24

MiRA Test-24 h

and LDMBB-24 h


MiRA Test-24 h

and LDMBG-24 h

0.484 Moderate agreement < 0.001

Both tests could provide practitioners and researchers quick and accurate methods of

detecting antibiotic residues in milk samples. However, the locally developed test

was cheaper than the commercial test (Table 4.11), and generally showed a clear

inhibition zone in the case of positive residues which could be easily recognized even

49

by inexperienced persons (Figure 4.6). The levels of agreements for other test pairs

were ranged from slight to moderate agreement.

Table (4.11): Comparative costing for conducting 50 tests by MiRA and

LDMBB tests

Name of test MiRA Test LDMBB

test

Content of the package 50 test 50 test

Cost 260 $ 30$

(A) LDMBB test (B) MiRA Test

Figure (4.6): LDMBB test (A) and MiRA Test (B)

Martin, Meek, & Willeberg, (1987) suggested that if kappa is high, the tests are

measuring what they purport to measure, but if it is low, much uncertainty exists, and

in order to confirm the results obtained by those tests more specific and sensitive

techniques should be used. Such techniques will also provide answers regarding the

sensitivity and specificity of the tests.

In this study however, neither sensitivity (also called the true positive rate, the

probability of correct identification of positive milk samples), nor specificity (also

called the true negative rate, the probability of correct identification of true negative

50

milk samples) were determined. Accordingly, the study does not claim that it

identified the proportion of positive samples that are really positive for antibiotic

residues, or the proportion of negative samples that are really negative residues.

4.9 Performance of B. subtilis vs. G. stearthermophillus on agar medium

Modification of the test by addition of agar to the locally developed medium for the

purpose of detecting antibiotic residues in fresh milk samples on solid media and for

comparing the performance of the isolated strain B. subtilis and that of the kit, G.

stearthermophillus was made without experiencing any difficulties in the application

of the test. The zones of inhibition were primarily measured in millimeter and then

re-categorized according to the size of inhibition zones as “very large”, “large”,

“small” and “no zone” of inhibition. They were also classified as positive and

negative, where samples exhibited clear zone of inhibition of more than 10 mm were

considered as positive otherwise they were negative. Table 4.12 compares the

frequencies and percentages of the four categories of zones of inhibition occurred at

agar plates by using B. subtilis and G. stearthermophillus after 24 h incubation

period.

Table (4.12): Frequencies and percentages (%) of zones of inhibition occurred

at agar plates made by locally developed medium by using B. subtilis and G.

stearthermophillus after 24 h incubation period

Zone size category B. subtilis G. stearthermophillus

No zone 13 (27.1%) 14 (29.2%)

Small 13 (27.1%) 19 (39.6%)

Large 12 (25.0%) 12 (25.0%)

Very large 10 (20.8%) 3 (6.3%)

Total 48* 48*

*: No bacterial growth was detected in 33 plates so they were excluded from

comparison.

While the percentages of milk samples exhibited “no zone” and “small zone” of

inhibition were less with B. subtilis than that of G. stearthermophillus, the

percentage of samples showed “very large zones” with B. subtilis was higher

51

(20.8%) than that of G. stearthermophillus (6.3%). The percentages of samples

exhibited large zone of inhibition by using B. subtilis and G. stearthermophillus

were the same (Figure 4.7)

Figure (4.7): Solid MRVP media showing zone of inhibition

On the other hand, using the Wilcoxon signed rank test to compare the patterns of

inhibition zones developed after 24 hours incubation for both strains (B. subtilis and

G. stearthermophillus) by using the same milk samples revealed a statistically

significant difference between the two test (p = 0.001). Of the 48 samples compared,

the positive ranks for the medium with B. subtilis was more than that of G.

stearthermophillus in 13 samples and similar results were obtained in 35 samples.

The comparison of the positive and negative results using Cohen’s Kappa illustrated

that there was almost perfect level of agreement (k = 0.948) between the tests using

the two microorganisms, B. subtilis or G. stearothermophillus.

The microbial growth inhibition is useful mechanism of antibiotic detection. One of

the advantage of microbial inhibition tests is that they target a broad spectrum of

antimicrobial compounds compared to family or antibiotic specific antibiotic binding

based tests such as those utilizing antibodies or other bacterial binders/receptors.

Differences in the results of the different microbial growth inhibition tests may be

due to differences in tests specificities which may depend on many parameters that

affect the test. Such parameters include growth ability of the organism used, amount

or concentration of growth organism or spores used, vessel dimensions, media

volume, media type, the nutrients mix provided, incubation time and the pH and

temperature used.

52

The test described in this study is very simple to carry out, do not depend upon

specialized equipment and the persons who perform the test do not have to be

trained. The result is very simple to determine. After the incubation period, the color

of the agar medium containing the indicator shows if test organism growth did occur

or not. Furthermore, microbiological screening methods are highly cost-effective

compared to physical or chemical detection methods.

53

Chapter V

Conclusions and

Recommendations

54

Chapter V

Conclusions and Recommendations

5.1 Conclusions

According to the findings of this study, the following conclusions could be drawn:

1- A total of 117 bacterial isolates that were recovered from 29 soil samples from

different areas.

2- Only those that showed high susceptibility to antimicrobials (34 isolation) were

selected for further testing.

3- Only one isolate (Isolate no. 25.5) was selected because it exhibited good

growth on both liquid and solid media.

4- Isolate no. 25.5 was identified as B. subtillis.

5- Indicator media composed of Buffered peptone 7 grams, Dextrose 5 grams,

Dipotassium phosphate 5, and (BromoCresol Purple) Indicator 0.03/litter in

broth media. For Agar media 15 grams per litter were added.

6- Only 49.4% of all milk samples showed total bacterial counts below the

allowable limits (Below 105 cfu/ml)

7- Using the commercial kit, great variation between the 4 hours (recommended

by the manufacturer) and 24 hours reading of the results was found. (4 hours =

23.5% Negative and 75.5% positive; 24 hours =82.7% negative, 17.3%

positive).

8- Using the locally developed kit, less variation between the hours and the 24

readings (4 hours =17.3% negative and 82.7% positive; 24 hours =64.2%

negative, 35.81% positive)

9- While the percentages of milk samples exhibited “no zone” (27.1%) “small

zone”(27.1%) and “very large zones” (25%) of inhibition in the solid media test

used B. subtillis .

10- While the percentages of milk samples exhibited “no zone” (29.2%) “small

zone”(39.6%) and “very large zones” (25%) of inhibition in the solid media test

used B. stearothermophilus

55

5.2 Recommendations

In light of the above conclusions and based on the results of this study, the following

recommendations are suggested

1- The results showed that a high percentage of milk contains antimicrobial

residues. Therefore, it is recommend that to the concerned authorities start

implementing control measures to prevent un-necessary use of antibiotics.

2- It is recommended to periodically check milk used in the industries to avoid

antimicrobial residues because of its harmful effects on the consumer.

3- The use of the developed kit in this study is recommended. It showed

comparable results to that of the commercially available kit aside from its low

cost.

4- Extend the testing scope of the newly developed kit to examine for antibiotic

residues in other products such as fish and meat.

5- Further research to improve the production of B. subtilis isolate as a step

toward commercialization of the locally developed kit.

6- Educational and awareness programs also should be implemented for

farmers, dairy producers as well as for the public on the risks of antibiotic

residues in milk.

7- Antimicrobial resistance for bacteria common between animals and human

should also be evaluated and monitored.

8- Withdrawal period of antibiotics should be observed when the animal

producing the milk has been given antibiotic for whatever reason.

9- Improving hygienic measures and standard precautions may reduce the need

for using antibiotics.

10- A more comprehensive and nationwide study investigating the presence of all

groups of antibiotics and a larger number of samples would be of a great

value

56

Reference

57

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61

Appendices

Appendix 1: Antibiotic susceptibility of strains isolated from soil sample

# Beta- Lactams Family Am

inoco

um

arin

Macro

lides

Chlo

ramphen

icol

Sulp

haam

ides

Flu

oro

quin

olo

ne

Tetracy

cline

Am

inogly

cosid

es

family

pen

icillin

Cep

halo

sporin

family

Isolate

Pen

icillin G

Am

oxicillin

Am

oxyclav

Clo

xacillin

Oxacillin

Cefalex

in C

efadro

ixl

Cefo

taxim

e

Cefazo

lin

Cefix

ime

Novobio

cin A

zithro

mycin

Ery

thro

mycin

Chlo

ramphen

icol

Co

-Trim

oxazo

le

Norflo

xacin

Tetracy

cline

Neo

myicin

Gen

tamicin

1 1,1 19 22 23 9 20 30 34 20 30 R 22 25 28 29 32 22 15 16 22

2 1,.2 16 20 19 R R 14 17 54 16 44 36 26 27 26 36 31 23 24 14

3 2,1 13 13 15 R R 9 18 14 8 R 18 R R 13 40 30 19 20 20

2 4 2,2 31 38 36 10 11 22 30 40 36 22 30 26 30 30 35 28 30 20 22

5 2,3 R R R R R R 13 R R R 20 22 26 23 16 25 24 20 20

6 2,4 34 30 32 9 12 36 34 24 22 R 26 32 32 32 40 32 34 22 25

7 2,5 25 27 24 R R 13 20 29 14 R 44 25 15 22 44 26 30 29 33

8 3,1 32 14 36 R R 24 30 38 32 22 32 26 34 30 36 30 34 20 24

9 3,2 26 29 26 12 12 30 29 22 26 R 26 26 30 29 36 28 30 21 23

10 3,3 11 11 11 R R R 22 10 13 R 27 24 27 27 R 27 26 19 22

11 3,4 16 17 16 R R 29 34 22 11 R 21 R R 17 40 30 21 20 18

12 4,1 26 22 30 R R 36 20 20 30 R 28 36 36 32 36 34 12 21 28

1 13 4,2 50 34 56 14 22 16 54 48 16 36 32 34 36 34 40 39 42 30 32

14 4,3 16 13 15 R R R 22 12 R R 21 R R 17 40 28 18 18 24

62

15 4,4 10 10 11 R R 18 22 10 R R 22 19 20 18 R 22 24 15 16

16 5,1 34 40 27 R R 28 26 R 28 R 24 24 26 19 46 32 30 21 19

17 5,2 21 26 20 7 18 28 30 30 26 18 20 24 24 27 26 19 18 R 8

18 5,3 R R R R R 8 13 R R R 24 19 21 21 R 23 23 18 19

19 6,1 25 24 30 8 9 32 18 26 30 R 28 32 36 32 38 34 37 21 22

20 6,2 12 11 17 R R 20 24 16 17 8 20 R R 11 36 22 19 19 21

21 6,3 R 9 R R R 15 18 9 R R 21 22 26 16 R 23 26 17 19

22 6,4 R 12 R R R R 17 9 R R 25 22 26 16 R 23 22 18 18

3 23 7,1 38 30 29 8 14 30 36 26 28 16 27 28 30 26 30 26 30 20 22

24 7,2 23 24 24 9 R 29 30 24 22 23 22 25 26 22 25 22 27 20 21

25 7,3 21 26 26 R 13 20 26 28 22 8 23 24 27 26 12 28 29 18 25

4 26 7,4 50 50 42 20 28 36 10 28 34 15 30 30 14 30 8 36 38 30 30

27 8,1 24 22 22 R 9 24 34 30 20 26 23 26 26 26 28 22 26 21 22

28 8,2 40 38 34 R 18 26 26 28 30 14 28 28 32 26 30 28 32 22 23

29 8,3 31 30 28 11 14 32 28 22 38 R 29 26 30 27 30 30 31 23 26

30 8,4 26 22 20 R R 32 34 24 24 16 30 28 30 23 32 32 33 20 20

31 9,1 R 16 14 R R 21 19 28 13 36 32 26 27 25 31 31 21 22 13

32 9,2 27 29 23 R 8 34 34 32 20 26 24 25 26 27 27 22 26 20 22

33 9,3 24 27 21 R 10 34 34 25 22 R 25 26 28 25 30 26 28 16 18

34 9,4 28 31 27 R R 32 32 R 24 R 22 28 30 28 44 28 26 21 23

35 9,5 12 14 11 R R 25 29 15 8 R 27 25 28 24 20 27 14 18 20

36 10,1 22 28 23 R R 11 17 22 11 R 30 24 14 15 38 30 35 25 29

37 10,2 R 18 14 R R 12 17 20 R R 20 R R R 30 24 23 R 22

38 10,3 15 19 18 R R 17 21 24 18 R 25 10 11 25 R 26 31 22 26

39 10,4 26 31 20 R R 11 20 26 14 R 30 28 16 21 40 28 34 23 30

5 40 11,1 34 30 28 13 10 36 36 16 16 16 23 15 24 30 39 32 29 23 30

41 11,2 27 28 24 R 10 36 36 36 12 20 28 28 22 32 32 30 33 20 26

42 11,3 24 30 22 R R 10 18 25 12 R 30 26 16 20 36 28 36 26 31

43 11,4 14 20 10 R R 20 30 20 20 R 23 R R 15 39 30 31 14 29

44 12,1 24 24 28 R 10 36 28 40 32 R 22 26 30 28 36 28 16 17 22

45 12,2 16 23 19 R R 8 16 21 11 8 25 26 26 11 30 25 28 21 34

6 46 12,3 30 24 24 8 14 36 34 30 26 24 26 24 26 25 25 22 32 25 26

47 12,4 30 22 25 11 12 32 32 30 34 R 21 26 28 30 33 33 26 22 26

48 12,5 17 15 18 R R 28 28 20 17 R 22 R R 16 30 28 19 17 25

63

49 13,1 19 23 20 R R 12 19 22 14 R 13 12 22 20 32 25 27 22 27

50 13,2 17 14 11 R R 18 20 34 13 R 20 7 R 17 31 26 26 20 25

51 13,3 20 21 19 R R 30 30 30 16 28 20 22 22 20 26 20 23 20 20

52 13,4 15 32 22 9 R 26 30 34 20 26 22 20 19 22 20 22 25 20 21

53 13,5 10 11 8 R R 19 19 13 9 R 17 R R R R 32 17 18 21

54 14,1 29 28 21 R 20 32 36 34 26 21 25 18 21 30 29 25 39 21 29

55 14,2 12 R R R R R R R R R 30 21 R R R 30 18 26 31

56 14,3 16 28 R R R 36 30 R 26 R 21 R R 13 40 32 28 16 27

57 14,4 22 22 17 R R 23 30 30 15 R 20 20 15 23 R 23 38 27 32

58 14,5 16 27 8 R R 25 25 12 28 16 21 29 25 16 42 18 27 22 26

59 16,1 18 26 18 R R 22 25 30 15 R 20 26 18 22 40 25 37 14 33

60 16,2 27 26 24 R 15 34 33 30 24 21 25 24 19 27 25 25 30 22 25

7 61 16,3 30 36 29 10 15 26 36 36 34 12 22 26 18 28 32 24 31 20 26

62 16,4 27 26 25 R 28 35 26 22 22 9 26 R 10 21 38 32 28 27 29

63 16,5 R 12 R R R 12 16 R R R 22 24 20 14 13 27 27 20 24

64 16,6 22 30 26 R R 15 20 26 12 R 30 29 22 16 39 28 18 22 29

8 65 16,7 30 15 26 15 16 40 36 20 30 12 24 22 20 30 30 19 29 19 21

66 16,8 R 16 18 R R 18 24 15 14 R 25 R R 12 36 18 32 25 18

67 17,1 24 22 20 R 8 31 42 44 20 13 24 25 10 25 R 20 32 42 35

68 17,2 22 24 34 R 16 46 34 12 30 R 30 26 22 31 36 30 18 29 30

69 21,1 R 11 R R R 12 13 R R R 21 25 18 12 R 26 23 20 24

70 21,2 21 25 21 R R 28 25 19 25 R R 22 R R 16 23 27 21 25

71 21,3 29 30 31 R 14 36 40 11 26 22 28 30 22 31 31 26 32 22 27

72 21,4 24 24 26 R 10 25 34 24 12 R 26 30 19 30 40 31 19 22 28

73 21,5 38 24 34 R 22 34 40 10 26 R 24 30 23 23 37 24 35 22 26

74 21,6 28 30 28 R 10 24 34 36 26 25 27 26 27 29 30 25 34 24 26

75 24,1 11 13 R R R 12 24 R R R 25 20 17 18 R 28 31 20 25

76 24,2 32 26 26 R 8 10 40 38 26 21 24 26 22 27 28 24 32 22 27

77 24,3 30 34 26 9 24 34 38 20 28 R 25 26 22 12 32 30 25 21 28

78 24,4 26 27 23 R R 26 33 32 18 R 26 24 10 9 22 28 12 25 32

79 24,5 R 9 R R R 17 24 11 R R 20 23 18 14 R 24 33 18 21

80 25,1 24 28 26 R 10 26 32 10 22 R 20 28 22 23 40 30 30 27 27

81 25,2 21 19 20 R R 16 22 21 18 R 40 R R 29 R 30 30 29 30

82 25,3 12 R R R R 15 28 19 R R 17 R R R 34 24 17 17 22

64

83 25,4 20 16 16 12 R 27 30 24 16 R 21 R R 15 36 29 30 20 22

84 25,5 25 24 24 R 44 32 30 18 22 9 22 30 20 35 35 30 30 26 25

85 26,1 R 20 20 R 14 38 50 10 30 R 30 28 20 34 40 30 20 28 30

86 26,2 17 17 16 R R 23 28 20 15 R 22 R R 12 36 30 40 19 25

87 26,4 R 27 R R 14 27 30 R 23 R R 20 R 21 R 20 30 R 20

88 26,6 32 29 R 9 16 R 38 38 24 R 20 32 19 30 34 30 17 25 25

89 26,7 10 15 10 R R 10 18 20 R R 20 18 R 10 40 30 16 24 24

90 26,9 30 20 24 17 30 40 20 20 30 R 28 30 23 32 40 28 22 27 30

91 26,10 20 14 15 14 R 22 22 15 13 R 20 R R 18 38 26 30 20 28

92 27,1 R R R R R 20 22 20 R R R 20 R 20 30 30 14 15 12

93 27,2 38 32 26 10 14 30 32 10 25 R 24 24 20 25 40 32 32 20 27

94 27,4 R 36 R R R R R R R R R 22 R 20 R R R R R

95 27,5 R 36 R R 26 27 18 20 34 R R 34 R 20 R 30 20 R R

96 27,6 23 28 24 7 R 11 17 24 10 R 30 24 18 28 32 28 30 36 26

97 27,7 22 19 22 R 10 10 16 19 21 R 21 16 21 33 38 30 35 28 30

98 27,8 38 20 30 10 R 26 36 R 20 R 20 10 19 22 36 20 23 26 29

99 27,9 33 30 27 15 16 40 40 20 40 R 24 28 20 30 30 30 20 22 25

100 27,10 26 20 R R 11 24 30 20 15 13 10 28 R 30 40 32 34 25 25

101 28,1 R 22 R R R 15 16 22 10 R R 22 R 20 24 25 21 15 R

102 28,2 36 40 30 R 10 20 20 26 20 R 22 36 22 30 20 13 27 27 30

103 28,3 50 40 R R 28 40 40 26 24 20 12 40 42 40 10 40 36 16 24

104 28,4 R R R R R R 12 R R R R 22 R 20 R 26 22 12 R

105 28,5 38 28 24 12 R R R 26 R 10 R 30 R 17 21 22 30 22 22

106 28,7 20 28 23 R 17 30 30 15 21 R 22 26 22 34 36 30 12 22 22

107 29,1 16 18 R R R 10 13 14 16 R 34 R 16 16 30 14 30 20 20

108 29,2 20 17 R R R 23 12 16 16 R 22 30 R 16 R 28 20 20 24

109 29,3 17 15 20 R R 20 23 17 14 R 27 27 18 16 38 30 27 19 24

110 29,4 42 22 40 10 20 30 30 40 36 14 38 34 22 18 40 30 42 24 30

111 29,7 24 28 R 18 14 40 36 20 30 16 23 18 24 30 19 13 34 20 20

112 29,9 26 20 23 R 9 30 30 30 20 15 19 19 13 26 28 21 22 23 23

113 29,10 14 15 11 R R R 26 20 10 R 25 R R 20 R 28 28 23 22

114 29,11 13 14 12 R R R R 36 R R 36 24 12 16 30 25 13 27 30

115 29,12 28 22 20 R 12 34 40 38 22 R 25 22 20 30 29 24 26 26 26

116 29,13 24 20 18 R R 10 20 28 14 R 23 20 R 25 25 23 30 25 20

65

R: mean no zoon .

* Antibiotic-impregnated discs (6 mm) with amount. in µg shown in brackets.

+ Diameter of inhibition from three individual experiments. S. sensitive; I. intermediate; R. resistant. Z:mean two zoon around antibiotic disc.

development of antibiotic detection bioassay kit from ... · ii abstract background: the presence...

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