Development of Antibiotic Detection Bioassay Kit from ... · II Abstract Background: The presence...
Transcript of 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.
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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
12 Rafah 31,267839 34,273337 27 KhanYounes 31,298810 34,355212
13 Rafah 31,267852 34,273290 28 KhanYounes 31,298724 34,354990
14 Rafah 31,267820 34,273266 29 KhanYounes 31,299200 34,355913
15 Rafah 31,267821 34,273267 30 KhanYounes 31,299261 34,355946
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
0.292 Fair agreement < 0.001
24
MiRA Test-24 h
and LDMBB-24 h
0.303 Fair agreement < 0.001
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.