Naruto Uzumaki Final Version of Thesis

Evaluation of Antibacterial potential of Moringa oleifera
Against the isolates of Diabetic foot Infection along with the

Profiling of Bacterial prevalence and Antibiotic resistance
among them
A thesis submitted
In partial fulfillment of requirements for the degree
Of
Bachelor of Science
(Microbiology)
By
Hamdan Aali
BS-M-18-28
Session: 2018-22
Institute Of Pure and Applied Biology
Bahauddin Zakariya University, Multan.
i
In the name of
Allah
The most merciful the most beneficent
“Praise be to Allah, Lord of the world, the beneficent, the merciful; owner of the
day of judgment. Thee (Alone) we worship, thee (Alone) we ask for help. Show us
the straight path, the path of those whom thou hast favored; not (the path) of those
who earn thin anger nor of those who go astray.”
(Surah al-fateha)
ii
INSTITUTE OF PURE AND APPLIED BILOGY, MICROBIOLOGY
DIVISION
BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN
Research Supervisor ___________________

Associate Prof. Dr. Mubashir Aziz
External Examiner ___________________
Director ___________________
Prof. Dr. Aleem Ahmad Khan
Research student ___________________

Hamdan Aali
Roll No: BMCBI-18-28
Viva Held on ___________________
iii
APPROVAL CERTIFICATE
The thesis entitled
EFFECT OF GRAPHENE OXIDE AND GALLIC ACID COMPLEX ON

WOUND HEALING IN DIABETIC RAT MODEL
Prepared by Hamdan Aali under my guidance is partial fulfillment of the

requirements for the degree of BS-Microbiology is hereby approved by
submission.
RESEARCH SUPERVISOR
___________________
Associate Prof. Dr. Mubashir Aziz
Institute of pure and applied biology
(Microbiology Division)
iv
Dedication
I dedicate my entire thesis work to my parents for their efforts in putting their
children to the pursuit of knowledge. Throughout my life, they have supported me
in my determination to find and realize my potential. Special thanks to my fellows
for their support and prayers.
v
Acknowledgement
In the name of ALLAH ALMIGHTY, the most gracious and the most merciful.
With great love and respect Durood-o-salam upon PROPHET MUHAMMAD
(PBUH). I don’t know how to choose diction to praise ALLAH ALMIGHTY, I
would say ALHUMDULILLAH for His great help that made me able to complete
my work successfully. This thesis presented by me is one of those countless
blessings that ALLAH ALMIGHTY, the praise worthy bestowed me. I thanked
ALLAH ALMIGHTY and myself fortunate at that day when my supervisor
selected me as his research student.
I would like to pay my respect to Associate PROF. Dr. MUBASHIR AZIZ. He is the
most devoted and competent person I have ever seen in my life. His
multidimensional personality was a guide for me throughout my research work.
Apart from his constant support during my research work, his high moral values
made him a role model for me. In short without his help, supervision and
enthusiastic attitude I would not be able to complete my task. I salute him with
great respect.
I would like to extend my appreciation to the Division of microbiology, IPAB.

Appreciation goes to the teaching and non-teaching staff of the division of
Microbiology whose encouragement contributed significantly to the successful
completion of my work. I am also thankful to Bilal Zahid and Aimen Fatima for
his great help and sincere support in completion of my work.
The initial source of inspiration that brought me to position where I stand

presently is circle of prayers and sacred wishes of my parents. While describing
their love, care, support and devotion, my diction is limited. Words lose their value
and feelings touch the peak of sentiment while expressing my acknowledgement
about my parents. The big credit goes to my friend Adnan Arshad, M. Mujahid,
Yousaf Sarfaraz, & Ayesha Latif for their love, source of help and inspiration at
every step during my research work. Thanks to ALLAH ALMIGHTY for giving
me His most valuable and great blessings in the form of my teachers, my parents
and friends.
Hamdan Aali
vi
CONTENTS
Sr. No Title Page No.
I. Abstract 1
II. Aims and Objectives 2
Chapter#1 Introduction
1.1 Introduction of Diabetes 3
1.2 Classification of Diabetes Mellitus 3
1.3 Epidemiology of Diabetes Mellitus 4
1.3.1 Prevalence of Diabetes Mellitus Type 1 4
1.3.2 Prevalence of Diabetes Mellitus Type 2 5
1.4 Insulin resistance and Hyperglycemia 6
1.5 Epidemiology and pathogenesis of complications 6
from Diabetes
1.5.1 Common mechanisms for Microvascular and 7
Macrovascular diseases in Diabetes
1.6 Hyperglycemia and susceptibility to infection 8
1.6.1 Impairment of Cytokine production 9
1.6.2 Leukocyte recruitment Inhibition 10
1.6.3 Defects in Pathogen recognition 11
1.6.4 Neutrophil Dysfunction 11
1.6.5 Macrophage Dysfunction 11
1.6.6 Natural killer cell Dysfunction 12
1.6.7 Inhibition of Antibodies and Complement effector 12
1.7 Healing in Diabetes 12
1.7.1 Factors involve in slow healing at cellular level 12
1.7.2 Metabolic deficiencies as a factor for stalled 13
healing
1.7.3 Altered miRNA level contributes in slow healing 13
1.8 Diabetic foot ulceration 14
1.8.1 Epidemiology of Diabetic foot infection 14
1.8.2 Pathophysiology of Diabetic foot infection 14
1.8.3 Types of Diabetic foot ulcers 15
vii
1.8.4 Bacterial Profile of Diabetic foot infection 15
1.8.5 Staphylococcus aureus 16
1.8.6 Escherichia coli 16
1.8.7 Pseudomonas aeruginosa 17
1.9 Moringa oleifera 17
1.9.1 Traditional medicine and research 17
1.9.2 Antibacterial activity of Moringa oleifera 18
1.9.3 Ant diabetic activity of Moringa oleifera 18
Chapter#2 Material and Methods
2.1 Materials 19
2.2 Methodology 19
2.2.1 Collection of Moringa oleifera leaves 19
2.2.2 Preparation of Ethanolic extraction 19
2.2.3 Media preparation 19
2.2.4 Preparation of swabs for sampling 20
2.2.5 Collection of samples 20
2.2.6 Sample enrichment 20
2.2.7 Subculturing 20
2.2.8 Identification and conformation of bacterial 21
species
2.2.9 Kirby Bauer Disc Diffusion Test 21
2.2.10 MIC and MBC assay 22
Chapter#3 Results
3.1 Immunological history of patients suffering DFIs 23
3.2 Bacterial Prevalence 24
3.3 Identification of Bacteria 24
3.3.1 Bacteria identification through culture media 24
3.3.2 Biochemical testing for Bacterial identification 25
3.4 Determination of Antibiotic Resistance profile of
Isolated bacterial species by Kirby Bauer Disk 28
Diffusion Test
viii
3.5. Determination of antimicrobial activity of
ethanolic leaf extract of Moringa oleifera by Agar 40
well diffusion method
3.6. Determination of MIC &MBC values of ethanolic 52
leaf extract of Moringa oleifera by 96-wells titer
plate method
Chapter#4 Discussion
4.1 Discussion 58
4.2 Conclusion 59
References
References 60
ix
List of Tables
r. No Title Page No.
1 Immunologic History of Patients with DFIs 23

2 Prevalence of Pathogenic Isolates from Diabetic 24
Foot Infections
3 Biochemical test of Bacteria 25
4 Antimicrobial Sensitivity Potential of different 28
antibiotics against Staphylococcus aureus
antibiotics against Pseudomonas aeruginosa
antibiotics against Salmonella typhimurium
antibiotics against Escherichia coli
antibiotics against Klebsiella pneumonia
antibiotics against proteus mirabilis
10 Antimicrobial potential of ethanolic extraction of 40
Moringa oleifera against Staphylococcus aureus by
agar well diffusion method (n=30)
Moringa oleifera against Pseudomonas aeruginosa
by agar well diffusion method(n=30)
Moringa oleifera against Salmonella typhimurium
Moringa oleifera against Escherichia coli by agar
well diffusion method(n=12)
Moringa oleifera against Klebsiella pneumoniae by
agar well diffusion method(n=6)
Moringa oleifera against Proteus mirabilis by agar
x
16 Determination of MIC and MBC values of Ethanolic 52
Extract of Moringa oleifera against Staphylococcus
aureus Strains
Extract of Moringa oleifera against Pseudomonas
aeruginosa Strains
Extract of Moringa oleifera against Salmonella
typhimurium Strains
Extract of Moringa oleifera against Escherichia coli
Strains
Extract of Moringa oleifera against Klebsiella
pneumoniae Strains
Extract of Moringa oleifera against Proteus
mirabilis Strains
xi
List of Graphs

antibiotics against Staphylococcus aureus(n=30)
antibiotics against Pseudomonas aeruginosa(n=30)
antibiotics against Salmonella typhimurium(n=24)
antibiotics against Escherichia coli(n=12)
antibiotics against Klebsiella pneumonia(n=6)
antibiotics against proteus mirabilis(n=6)
Moringa oleifera against Pseudomonas aeruginosa
Moringa oleifera against Salmonella typhimurium
Moringa oleifera against Escherichia coli by agar
xii
List of Figures
1 Growth of Staphylococcus aureus on MSA 26
2 Growth of Salmonella typhimurium on EMB agar 26
3 Growth of pseudomonas aeruginosa on PCA 26
4 Growth of Escherichia coli on EMB agar 27
5 Growth of Klebsiella pneumoniae on EMB agar 27
6 Growth of Proteus mirabilis on MacConkey agar 27

antibiotics against Staphylococcus aureus(n=30)
antibiotics against Pseudomonas aeruginosa(n=30)
antibiotics against Salmonella typhimurium(n=24)
antibiotics against Escherichia coli(n=12)
antibiotics against Klebsiella pneumonia(n=6)
antibiotics against proteus mirabilis(n=6)
Moringa oleifera against Pseudomonas aeruginosa by
Moringa oleifera against Salmonella typhimurium by
Moringa oleifera against Escherichia coli by agar well
diffusion method(n=12)
xiii
xiv
Abstract
Foot infections are the major complications of diabetes mellitus and lead to the development of
gangrene and ultimately amputation of the limb. Proper diagnosis of the causative agents and
their antibiotic susceptibility play a significant role in preventing adverse prognosis of diabetic
foot. 30 patients having diabetic foot ulcers of Wagner’s Grade 1 or above were included in this
study. Debrided tissue, pus, or swabs from the base of the ulcers were subjected to aerobic
culture. The organisms were identified, and further antibiotic sensitivity of the aerobic bacteria
was conducted by Kirby–Bauer’s disc diffusion method. The antibacterial activity of ethanolic
extract of moringa were calculated against the extracted pathogens by agar well methods along
with tits MIC and MBC determined using 96 wells titer plates.
Thirty aerobic bacteria were isolated from these ulcers; The antibiotic resistance pattern of our
isolated pathogens reported in our study was as followed: (100%) & (89%) isolates of S. aureus
were resistant to Oxalic Acid and Fuscidic acid respectively while (78%) were sensitive to
ciprofloxacin and (21%) to Clindamycin (100%). Isolates of P. aeruginosa showed resistance
against Doripenem, Ticarcillin and Linezolid While some strains showed sensitivity to Colistin
(10%) and Clindamycin. S. typhimurium isolates were resistant to Ampicillin (100%), Linezolid
(94%) & Streptomycin (100%) respectively. While sensitivity was shown against Vancomycin
(10%) and Clindamycin (10%) by S typhimurium isolates. Strains of E coli shown (100%)
resistance against Ampicillin and Streptomycin, while they were (16%) & (25%) sensitive to
Vancomycin and Clindamycin respectively.in case of Klebsiella pneumoniae (100%) strains
were resistant to Ampicillin and Streptomycin while, (33%) & (66%) of K. pneumoniae strains
were susceptible to Colistin and Clindamycin. All (100%) strains of Proteus mirabilis shown
resistance for Streptomycin, Linezolid, and Ampicillin and among them (50%) & (49%) were
sensitive for Clindamycin and Vancomycin.
The finding shows that Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella

typhimurium, Escherichia coli, Klebsiella pneumonia, & Proteus mirabilis were the most
prevalent bacterial species in Diabetic foot infection. The bacterial strains were multidrug
resistant which leads to further complications, and infection become chronic and difficult to be
healed. Bacterial inoculums were collected to check the effect of Moringa oleifera. Moringa
oleifera compound as antimicrobial potential against all the bacterial isolates. Present study
reports that Moringa oleifera had effective antimicrobial potential against multidrug resistant
strains of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium,
Escherichia coli, Klebsiella pneumonia, & Proteus mirabilis and may have a promising effect in
controlling the Diabetic foot infection and to tackle the slow healing.
1
Aims and Objectives
 To study various epidemiological factors responsible for causing diabetic
foot infections.
 To study prevalence of major pathogens responsible for causing Diabetic
foot infections.
 To evaluate antibiotic resistance among pathogens isolated from diabetic
foot infection.
 To determine the extent of antibacterial activity of Ethanolic extract of
Moringa Oleifera against the isolated pathogen by Kirby Bauer Diffusion
method along with the evaluation of its MIC & MBC values
2
Chapter No. 1
1.1. INTRODUCTION
Diabetes mellitus is a serious disorder that involves the metabolism of carbohydrates, fats and
proteins. One of the characteristic features of diabetes mellitus is a defective insulin secretory
response. The deficient or disordered protein secretion translates into impaired use of
carbohydrates, specifically glucose which results in hyperglycemia. (Kumar et al., 2020).
Diabetes mellitus is the most common endocrine disorder which is referred as “sugar” in local
language. The disease occurs due to deficiency or absence of insulin. Rarely, insulin resistance
also leads to diabetes mellitus. (“Ross & Wilson Anatomy and Physiology in Health and
Illness - 13th Edition,” n.d.). According to an estimation by The International Diabetes
Federation (IDF) there are over 19 million diabetic subjects in Pakistan.
Pancreas in our body releases both insulin and glucagon hormone. There are two types of cells in
Islets of Langerhans’; (ß) cells and alpha (α) cells. The alpha cells secrete glucagon and the beta
cells secrete insulin. Insulin decreases the level of glucose in blood through glucogenesis. It also
transports glucose into muscles, liver and adipose tissue. Erythrocytes and neural cells do not
require insulin for utilization of glucose. Alpha cells, on the other hand, play an important role in
maintaining blood glucose levels by accelerating the rate of glucogenesis. (Kumar et al., 2020).
Being a chronic disease, diabetes increases the potential risk of microvascular and macrovascular
damages to occur. It has negative impact on various organs including brain, kidney, heart and
eyes which increases the risk of several other diseases. Moreover, diabetic patients are more
susceptible to infections.
People with diabetes have a higher risk of adopting lower respiratory tract infections such as
pulmonary tuberculosis and pneumonia, urinary tract infections, and skin and soft tissue
infections. Moreover, diabetes also lowers the outcome of the infection treatment. The major con
of diabetes is that it increases economic burden on diabetic patients due to the high cost of care,
the length of treatment, and related complications (Afiat Berbudi et al., 2020).
1.2. Classification of Diabetes Mellitus
WHO published the first ever most accepted classification of diabetes mellitus in the year 1980,
The classification was modified in 1985. (Verge et al., 1996). The primary form of diabetes also
known as idiopathic diabetes is quite different from the secondary diabetes mellitus. In
secondary diabetes mellitus, certain forms of hyperglycemia occur which is associated with the
identifiable causes. These causes include the destruction of pancreatic islets induced by the
inflammatory pancreatic diseases, surgery, certain drugs, overloaded iron (Hemochromatosis),
tumors and various genetic and acquired endocrinopathies. (Kumar et al., 2020).
3
The classification includes clinical stages as well as etiological types of diabetes mellitus. It
encompasses other categories of hyperglycemia. (De Fronzo et al., 1992) . various factors play
their role in deciding what type of diabetes the suspect is suffering from. the circumstances at the
time of diagnosis have the major impact in assigning a certain type of diabetes to an individual. It
is not easy to fit many diabetic patients into the same category. (Lillioja et al., 1993).
Primary diabetes is a group of heterogenous disorders where hyperglycemia is a common

symptom in all of them. (Kumar et al., 2020). The terms of insulin-dependent (IDDM) and
non-insulin-dependent (NIDDM) were proposed by WHO in 1980 and 1985. These terms have
long disappeared and now we have the new terms of classification. The new classification
system has four types of diabetes mellitus: type 1 (IDDM), type 2 (NIDDM), other specific types
and gestational diabetes. The subsequent International Nomenclature of Diseases (IND) reflected
these types in 1991. The tenth revision of the International Classification of Diseases (ICD-10)
recognized these types in 1992.
1.3. Epidemiology of Diabetes Mellitus
The rate of diabetes is increasing day by day in every country. The major reason behind such
high rates of diabetes is global rise in the prevalence of obesity and unhealthy lifestyles.
According to a latest estimation, about 382 million people had diabetes in 1023 and the rate was
expected to rise to 592 million people by 2035(Forouhi and Wareham, 2014).
The two main types of diabetes are Type 1 and Type 2. Type 2 diabetes accounts for the majority
(>85%) of total disease prevalence. However, both types of diabetes can lead to various
multisystemic complications of microvascular endpoints that include retinopathy, nephropathy
and neuropathy, and the macrovascular endpoints that include ischemic heart disease, stroke and
peripheral vascular disease.
Diabetes is an important public health concern as it has ever-lasting effects like morbidity,
mortality, reduced life expectancy and financial burden due to costs of diabetes.
1.3.1. Prevalence of Diabetes Mellitus type 1
Type 1 diabetes can occur at any age but the records show the highest chance of occurrence of
disease from birth till 16 years of age. However, almost all populations show a sharp increase of
incidence rate in age group around 10 – 15 years. the population-based incidence rate was not
found in people of ages above 35 years. In more incidence countries, the rate of diabetes is
higher in male children comparatively. The rate was seen opposite in lower incidence countries.
However, the differences are very small. (DIAMOND Project Group, 2006).
In young adults, there is more excess in males generally, (Gale and Gillespie, 2001) and in most
populations, the peak incidence is around puberty. The chances of type 1 diabetes fluctuate
nearly 400 times in children of different countries. The age-adjusted incidence rates range from
4
0.1 per 100,000 annually in some parts of Venezuela and China to 37.8 per annum in Sardinia
and 40.9 per 100,000 yearly in Finland (DIAMOND Project Group, 2006) .
In several other countries including China the incidence varies by a 12-fold variation per year in
Europe as per report. (Patterson et al., 2009) . it is noticed that the relative magnitude of
increase in incidence is much greater in low-incidence countries. The most noticed increase is
seen in the youngest age group (0 – 4 years). season is also a important factor that affects the
incidence of type 1. The incidence becomes highest in autumn and winter. (Moltchanova et al.,
2009).
1.3.2. Prevalence of Diabetes Mellitus type 2
The incidence of diabetes type 2 in Europe is around 7 per 1000 annually in Western populations
(Forouhi et al., 2007). In people with IGT (Impaired glucose tolerance), the incidence is
reported to be about tenfold greater than in individuals with normal glucose tolerance. (de Vegt
et al., 2001).
The risk of diabetes progression in future is also greater in people who have other hyperglycemic
states including gestational diabetes mellitus. The US Centre for Disease Control reported the
data which showed the increase in rate of diabetes. The number of diagnosed diabetic cases was
quadrupled from 5.5 million person in 1980 to 21.1 million people in 2010. This increase was
mainly a sign of rapid increase in obesity among people. there is a sharp increase in the
prevalence of diabetes globally. It is projected to increase from 382 million (8.3%) in 2013 to
592 million (10.1%) in 2035.
In developing countries, there is even more estimation of prevalence of the disease. In the areas
where populations tend to adopt western lifestyle, the incidence of diabetes is increasing very
rapidly. (Dabelea et al., 2014). There has been rapid increase in the spread of obesity among
children from very young age. It has led to the prevalence of type 2 diabetes in children and
young adults specially in people who belong to susceptible ethnic groups.
The rate of diabetes type 2 was seen to be much higher in American, Indian, black and Hispanic
youth as compared to the white youth in the United States. This rate increased overall from 0.34
per 1000 (95% confidence interval 0.31–0.37) in 2001 to 0.46 per 1000 (95% CI, 0.43–0.49) in
2009.
Generally, prevalence rate was lowest n the rural areas of developing countries, intermediate in
developed countries. This rate was seen to be highest in certain races who have adopted the
Western lifestyle or tend to adopt the patterns. The prevalence is higher in people who have
higher prevalence of obesity. There is a hypothetical concept that genetic susceptibility to obesity
is disadvantageous in the case of food abundance but it turns out to be advantageous in case of
food scarcity. So, the persistence is driven by natural selection. The evidences of gene-
environment interaction support this hypothesis of “thrifty genotype”.
The individuals who move from rural or low-prevalence areas to the west are at a very high risk
to adopt type 2 diabetes. In UK, diabetes is four to six times more prevalent in South Asians and
African Caribbeans as compared to the European white population. (Forouhi et al., 2007).
5
Globally, there is not a huge gender difference in the number of people having diabetes.
According to an estimation in 2013, there are 14 million more men having diabetes than women.
However, the incidence of spread of diabetes increases sharply with age in both genders.
1.4.Insulin Resistance and hyperglycemia
Insulin production in induced by increased blood glucose levels after eating. The secretion by the
islet β cells into the blood also increases after eating. Insulin binds with insulin receptors in the
cell membrane and induces the translocation of glucose transporters towards the cell membrane.
As a result, the glucose uptake by the cells increases and there is decreased level of glucose in
the blood. Hyperglycemia is a result of failure of pancreas to produce the sufficient amount of
insulin, improper insulin action or both of them. In long term, hyperglycemia damages or fails
various organs and tissues of the body. In mice modeling, it was noticed that obese mice with
elevated levels of tumor necrosis factor (TNF)-α in the adipose tissue were found to be insulin
resistant. Moreover, the levels of interleukin (IL)-6, plasminogen activator inhibitor, C-reactive
protein and various other inflammation mediators were hoisted. TNF-α, free fatty acids,
diacylglyceride, ceramide, reactive oxygen species (ROS), hypoxia activate Iҡβα kinase β
(IKKβ), and c-Jun N-terminal kinase I (JNK1) in adipose tissue and the liver induce insulin
receptor substrate (IRS-1) inhibition. Insulin resistance is also triggered by TNF-α by inhibiting
the peroxisome proliferator-activated receptor-gamma function.
Tyrosine phosphorylation occurs at IRS-1 and IRS-2 as insulin binds with its receptor. IKKβ and
JNK-1 are the mediators of stress and inflammatory response. These mediators inhibit the insulin
signaling due to the serine phosphorylation of IRS substances. Moreover, the transcriptional
activation of various genes related to inflammatory response is induced by JNK1 and IKKβ
which results in insulin resistance.
The signaling pathways of JNK1 and IKKβ are activated during obesity through the influx of
free fatty acids and glucose. Activated IKKβ phosphorylates Iκβα and promotes ubiquitination. It
also causes the degradation of Iκβα in proteasome which results in the translocation of NFκβ into
the nucleus and induces the transcription of various genes that are involved in inflammation.
1.5.Epidemiology and Pathogenesis of complications from diabetes
Diabetes is a serious disease that can affect various organs and organ systems of your body. It
can lead to some serious complications overtime. Mainly, the complications of diabetes are
divided into two types: macrovascular and microvascular. Macrovascular diabetes involves
bigger problems like cardiovascular disease, stroke and peripheral vascular disease. The
peripheral vascular disease can lead to bruises and injuries that often do not heal. It can cause
gangrene and ultimately lead to amputation. Microvascular complications include the damage of
nervous system (neuropathy), damage to the renal system (nephropathy) and eye damage
(retinopathy) according to the American Diabetes Association, 2006.
6
Microvascular complications in diabetes type 2 are the most reported cases in prevalence of
diabetes. Recent researches indicated that chronic kidney disease (microalbuminuria), foot
problems (foot lesions, numbness, foot/toe amputation) and eye damage (diabetes affects the
eyes and retinopathy happens). Macrovascular complications (heart attack, chest pain, coronary
heart disease, stroke and congestive heart failure) are comparatively less occurring.
Complications can be episodic (foot ulcers and infections) which can occur many times and can
be treated. They can also be progressive (nephropathy) that begin mildly and result in further
damage to the organ with time. Progressive complications can cause greater loss of functionality
which can neither be reversed nor treated properly.
Various other complications include dental problems, reduced infection resistance (influenza and
pneumonia), macrosomia and other birth complications in pregnant women having diabetes. the
types of complications are same for both the types of diabetes but the frequency and timing of
the occurrence of a complication can vary in type 1 and type 2 diabetes. The types and
prevalence of the most common diabetes complications are discussed further in more detail with
specific attention to differences between complications of type 1 versus type 2
diabetes.(Deshpande et al., 2008)
1.5.1. Common mechanisms for microvascular and macrovascular diseases in diabetes
Certain chemical reactions between the by-products if sugar and proteins occur over the course
of days and weeks. these reactions are the common pathogenic mechanisms for microvascular
disease as they produce irreversible cross-linked protein derivatives called AGE (Brownlee et
al., 1988). These derivatives leave a lasting effect on the surrounding tissues which include
modifications like thickening of collagen and endothelium.
In diabetic retinopathy, AGE can lead to the inhibition of growth and programmed cell death of
retinal pericytes (Yamagishi et al., 1995). It can induce over-production of endothelial growth
factors (including vascular endothelial growth factor, insulin-like growth factor 1, hepatocyte
growth factor and basic fibroblast growth factor) and increase pathologic angiogenesis also
known as neovascularization. It can also increase vascular inflammation and all of these
conditions lead to an increased risk for the formation of micro-thrombosis, blockage of the
capillary and retinal ischemia. (Boeri et al., 2001)
The diabetic persons might go through retinal fibrosis and detachment and loss of vision because
the neovascularization, vitreous hemorrhage and increased levels of vascular endothelial growth
factors lead to these conditions. AGE can also bind to immunoglobulin protein receptor for
AGE. This binding produces a cascade of signaling events that can lead to endothelial cell
dysfunction.
Advanced glycation end products also play an important role in DN through AGE-mediated
programmed cell death of mesangial cells. The altered extracellular matrix proteins appear to
contribute to both glomerular hyperfiltration and sclerosis. The formation of AGE can sometimes
7
stimulate the over-secretion of growth factors such as insulin-like growth factor 1 and
transforming growth factor β. They can further contribute to glomerular fibrosis, which leads to a
decrease in available glomerular surface area for filtration (Osterby et al., 1988) .
Other mechanisms involved in microvascular disease include the PKC pathway (a family of
multifunctional enzymes involved in signal transduction and gene expression of growth factors
and inflammatory signals (Spranger et al., 2001) that may increase vascular permeability) and
the polyol pathway (the enzymes aldose reductase and sorbitol dehydrogenase, which catalyze
reactions that can lead to sorbitol accumulation-associated osmotic and oxidative stress damage
to the endothelium). Peripheral nerve damage in DN, including neuronal degeneration and
impairment of regeneration of thinly myelinated fibers, is also mediated by AGE accumulation,
the activation of the polyol and PKC pathways, and the deleterious effects of ROS.
The RAS (Gilbert et al., 2003) can alter the glomerular hemodynamics and mediate sclerosis in
DN so it has an important role in the occurrence of microvascular disease. Some other enzymatic
pathways are also involved in the development and further progression of the microvascular
disease. However, we are not discussing them in detail here. Various factors have their role in
the pathogenesis of macrovascular disease in diabetes. the most common site of injury is the
vascular endothelium. At first, diabetes impairs the vasodilating ability of vascular endothelium
by inhibiting the nitric oxide pathway (NO, a gas molecule that maintains arteriolar
vasodilation). (William et al., 1996). Hyperglycemia inhibits the enzyme that produces NO
(endothelial nitric oxide synthase, [eNOS]) increases the production of ROS and leads to further
inhibition of eNOS. The vascular endothelium loses its ability to produce NO-activated tissue
plasminogen activator. It is a fibrinolytic (anti-clotting) protein that inhibits the ability of
inflammatory cells to “stick” to the endothelial surface (Grant, 2007). Insulin resistance induces
the decrease in NO production and leads to impaired vasodilatory response.
The adipose tissue starts to increase the release of free fatty acids(Hennes et al., 1996) which
triggers the PKC pathway. The PKC pathway can directly or indirectly inhibit the activity of
eNOS through the increased production of ROS (Inoguchi et al., 2000). The production of AGE
from hyperglycemia inhibits the production of NO which impairs the vasodilatory response in
diabetes. (Linden et al., 2008).
Besides the delay in vasodilatory response, the vasoconstriction is increased through the
overproduction of vasoconstrictor substances. These substances include endothelin 1 that has
direct vasoconstrictive effects on the endothelium and indirect effects on the fluid volume which
include stimulation of water and salt retention and RAS activation. (Rask-Madsen and King,
2007).
1.6.Hyperglycemia and susceptibility to Infection
The human body uses astonishing ways and amazing mechanisms for protection against the
invasion of innumerable bacteria, viruses, toxins, fungi and parasites. When the circumstances of
8
body are normal, the pathogens find it difficult to penetrate the defense system. However, several
conditions lead to the improper working of the immune system. For example, if you have an
open wound on your body, the bacteria can easily enter the body and cause infection which
produces pus.
Natural barriers (intact skin, mucosal surfaces) in the body, production of reactive oxygen
species, cytokines and chemokines defend the entry of pathogens in the immune system. In case
of diabetes, the immune system of the host is immunocompromised. The cellular immunity is
also affected by the negative effects of neuropathy and T2D. neuropathy can damage the natural
barriers. Hyperglycemia and insulin deficiency can lead to these conditions.
According to the American Diabetes Association, people suffering from diabetes should take
infections as a serious issue because of their weak or almost failed immune system. Various
studies have shown that diabetes-related mechanisms impair the defense system of host against
pathogens. These mechanisms include suppression of cytokine production, defects in
phagocytosis, dysfunction of immune cells, and failure to kill microbials.
1.6.1. Impairment of Cytokine Production
An in vitro study showed that peripheral blood mononuclear cells (PBMCs) and isolated
monocytes of individuals having T1D and T2D secrete less interleukin 1 beta (IL-1β) as
compared to controls after stimulation with lipopolysaccharides (LPS).
Another study demonstrated that monocytes isolated from PBMCs of T1D subjects secreted
lower IL-1 and IL-6 compared to healthy donors. PBMCs obtained from non-diabetic people
stimulated by anti-CD3 antibodies that were exposed to high glucose levels showed suppression
in the production of cytokines IL-2, IL-6, and IL-10. Since IL-6 is important for protection
against pathogens and for adaptive immune response by inducing antibody production and
effector T-cell development, these studies revealed that the immune response against invading
pathogens can be suppressed by the inhibition of cytokines in hyperglycemia.
Spindler et al. reported that reduced expression of IL-6 and IL-17A was shown in PBMCs
obtained from healthy subjects induced with dextrose octreotide. The expression was seen much
reduced especially in CD14+ and CD16+ intermediate monocytes. This indicates impaired
immune responses due to high blood glucose levels.
Some studies by Price et al. have shown that a loss of IL10 secretion by myeloid cells occurs due
to increased glycation. The study also demonstrated that there is a reduced production of
interferon gamma (IFNγ) and TNF-α by T cells. In addition, in comparison to normal mice, the
IL-22 cytokine is lower in high fat diet-induced hyperglycemic and obese leptin-receptor-
deficient (db/db) mice.
9
One of the recent studies by Hu et al. showed that type 1 IFN production is suppressed in high
glucose medium cultured PBMC which is stimulated by poly I:C.
Another study by Tan et al. demonstrated that in contrary to normal healthy donors, there is
lower production of IL-12 and IFNγ in PBMC cultures from diabetic subjects following
Burkholderia pseudomallei infection. Moreover, intracellular bacterial load was higher in
PBMCs of diabetic subjects compared to healthy controls. It clearly suggests that hyperglycemia
impairs the host’s defense against invading bacteria. The addition of recombinant IL-12 and
IFNγ worked efficiently against the bacterial load in PBMCs of diabetic subjects and reduced it
to some extent. The reduced amount indicates that the immune cells’ capacity to control bacterial
growth is impaired in diabetic patients due to low production of IL-12 and IFNγ. Therefore, it is
thought that hyperglycemia in diabetics attenuates the activity of macrophage and other
leukocytes in the elimination of pathogens.
The impact of insulin deficiency in T2D on macrophage activity against pathogens has not been
studied in detail unlike the effect of hyperglycemia on immune cell activity in T2D. A study by
Tessaro et al. regarding the impact of insulin deficiency on immune response, demonstrated that
the production of TNF-α and IL-6 after LPS stimulation increased significantly after the
administration of insulin into bone marrow-derived macrophages that were isolated from diabetic
mice.
Another study using rats revealed that lack of insulin resulted in a disruption in phagocytosis of
alveolar macrophages as well as cytokine release. However, insulin intervention restored both of
them. Since TNF-α and IL-6 play a role in leukocyte function against pathogens, this result
indicated that the administration of exogenous insulin in diabetes may enhance immune cell
activity and protect the body against pathogenic entry and infections.
1.6.2. Leukocyte Recruitment Inhibition
It was seen that the infiltration of CD45+ leukocytes and CD8+T cells reduced significantly in
the brains of db/db mice that were infected with West Nile virus-associated encephalitis. This
study showed that the attenuated expression of cell adhesion molecules (CAMs) such as E-
selectin and intracellular adhesion molecule (ICAM)-1 is somehow co-related with the
impairment of recruitment of CD45+ leukocytes and CD8+T cells.
In their in vivo study, Martinez et al. demonstrated the same defect in leukocyte recruitment
using streptozotocin-induced diabetic mice infected by Klebsiella pneumoniae. There were lower
number of granulocytes in the alveolar airspace of the diabetic mice. They also reported reduced
cytokine production—such as CXCL1, CXCL2, IL-1β, and TNF-α—in lung tissue following
lung exposure to K. pneumoniae LPS.
10
1.6.3. Defects in Pathogen Recognition
The detailed study by Martinez et al. reported that diabetic mice had reduced expression of Toll-
like receptor (TLR)-2 and Toll/IL-1R domain-containing adaptor protein (TIRAP), which play
role in pathogen recognition. However, several studies have shown increased expression of TLRs
in neutrophils and monocytes isolated from people with diabetes.
There were major differences among different categories of diabetics. An analysis by Gupta et al.
revealed that TLR expression was related to complications and glycemic conditions of the body.
It was lower in diabetic subjects with poor glycemic control and complications. However, they
were elevated in patients with well-controlled hyperglycemia without complications. Hence,
there is nothing clear about the impact of hyperglycemia on TLR expression and related
immunity in diabetic subjects.
1.6.4. Neutrophil Dysfunction
ROS product of insulated neutrophils from T2D tuberculosis cases following phorbol 12-
myristate 13- acetate stimulation was reduced. This disfigurement in ROS product was
associated with increased situations of resistin in T2D cases’ serum. Perner etal. reported
repression of superoxide (O2-) in insulated neutrophils from healthy subjects when they were
exposed to a high glucose attention medium. This impairment passed via glucose- 6- phosphate
dehydrogenase (G6PD) inhibition, which disturbed the conformation of nicotinamide adenine
dinucleotide phosphate (Perner et al., 2003).
Stegenga et al. induced hyperglycemia in the blood of healthy individuals and then challenged it
with bacterial wall components. As a result, there was a lower neutrophil degranulation in the
blood. Neutrophil dysfunction in phages S. aureus was also demonstrated due to C3-mediated
complement inhibition caused by hyperglycemia (Hair et al., 2012) .
In line with those studies, Joshi et al. reported that hyperglycemia led to suppressed activity of
neutrophil to produce neutrophil extracellular traps (NETs). This led diabetics to be more
susceptible to infections (Joshi et al., 2013) . All of these studies revealed that hyperglycemia
causes neutrophil dysfunction. The main dysfunction includes defects in ROS production (Chao
et al., 2015) , neutrophil degranulation impairment (Stegenga et al., 2008) , inhibition of
immunoglobulin-mediated opsonization , decreased phagocytosis, and NET formation defects
(Joshi et al., 2013) .
1.6.5. Macrophage Dysfunction
Hyperglycemia alters the function of macrophages as well. It was demonstrated that chronic
hyperglycemia was significantly associated with defects in Fcγ receptors and complement
receptors on isolated monocytes. This results in phagocytosis impairment (Restrepo et al.,
2014). A reduced anti-bacterial activity and phagocytosis was observed during an in vitro study
11
using macrophages derived from mice bone marrow and treated with high glucose (Pavlou et al.,
2018). A reduced phagocytic activity was also observed in peritoneal macrophages from diabetic
mice. This could be related to the reduced glycolytic capacity and reserve of macrophages that
can lead to long-term sensitization to high levels of glucose.
Another study used resident peritoneal macrophages (RPMs) isolated from mice. In this study,
Liu et al. demonstrated significantly reduced phagocytic activity and adhesion capacity in RPMs
of db/db mice (Liu et al., 2012). They also reported increased macrophage polarization shifting
to M2 macrophages in db/db mice as compared to normal mice used as control. Similarly,
increased M2 macrophage markers, including Arginase 1 and IL-10 were observed in
macrophages derived from mice bone marrow that were exposed to high glucose for a long
period of time (Pavlou et al., 2018). As a result, we can say that M2 macrophages have poor
microbicidal capacity. This shifting can weaken their immune response against bacterial
infection.
1.6.6. Natural Killer Cell Dysfunction
Natural killer cells (NK) are important for controlling invading pathogens. A study demonstrated
dysfunction of natural killer (NK) cells (Berrou et al., 2013) . In this study, isolated NK cells
from T2D subjects demonstrated defects in the activating receptors of NK cell. The receptors
NKG2D and NKp46 were associated with functional defects in NK degranulation capacity.
1.6.7. Inhibition of Antibodies and Complement Effector
An animal study showed the dysfunction of complement activation in rats (Mauriello et al.,
2014). They demonstrated that hyperglycemia was associated with decreased C4-fragment
opsonization, which inhibits classical or lectin pathways of complement activation.
1.7.Healing in Diabetes
Diabetes delays the process of healing. It leads to a non-healing wound including various
complications along with psychiatric stress and depression. These complications include
functional limitations, difficulty in walking, and infection like cellulitis, septicemia. abscess,
gangrene, and osteomyelitis. The healing process makes it necessary for the inflammatory cells
to collaborate with the biochemical mediators. The process should be stimulated by various
factors. However, alteration of the cellular and biochemical factors and various activities have
been involved in the failure of wound healing in diabetics.
1.7.1. Factors involve in slow healing at cellular level
Neutrophils, monocytes, macrophages, keratinocytes, fibroblasts, T cells, B cells, mast cells and
endothelial cells are the major cells involved in wound healing process. These cells are actively
involved in the production and regulation of various growth factors and cytokines. Monocyte is
the foremost producer of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and VEGF, IGF-1 and
12
TGFβ in both diabetic and non-diabetic conditions. Monocyte transforms into macrophages later
on. TNF-α, IL-10 and other cells, keratinocytes, fibroblasts, mast cells and endothelial cells are
also significantly produced by Neutrophils along with T and B cells. These cells also contribute
in production of VEGF, IGF-1 and TGF-β. Macrophages play a crucial role in the process of
healing. Hyperglycemia and oxidative stress change the epigenic code that results into change in
macrophage polarization and its modulation. One of the main reasons that delays wound healing
is dis-regulated macrophage polarization. Studies revealed that in diabetes, a complex
mechanism is responsible for delay in wound healing at molecular level. Activities like sustained
production of pro-inflammatory cytokines, impaired angiogenic response and microvascular
complications, impaired macrophage and neutrophil’s function. They also impaired keratinocytes
and fibroblast migration and proliferation. Impaired production of healing-associated factors like
impaired growth factor production has been reported in diabetic animal models.
1.7.2. Metabolic deficiencies as a factor for stalled healing
The phases of healing process in diabetic patients are affected by various factors including
specific metabolic deficiencies, impaired physiological responses like hypoxia due to glycation
of hemoglobin, alteration in the membranes of red blood cells and narrowing of blood vessels. In
hypoxia, there is decreased oxygen supply to the wounds because the blood vessels are
narrowed. The healing process is further delayed due to glycation of hemoglobin which causes
deficient supply of nutrients and oxygen to the tissue. Hypoxia/glucose deficiency and deformed
proteins produce a stress response to the cell by accretion of unfolded proteins within
endoplasmic reticulum known as UPR. This UPR activates right after the tissue or skin gets
injured. It induces the production of pro-inflammatory mediators. DWs showed sustained
induction of UPR along with augmented expression of the pro-inflammatory chemokine as
compared to normal wounds.
1.7.3. Altered miRNAs level contributes in slow healing
Wound healing in diabetes also delays due to microvascular complications causing local
ischemia. miRNAs9 are a class of noncoding RNAs of 19–24 nucleotides that link in numerous
physiological processes. This interlinking plays a very crucial role in microvascular
complications. Different levels of miRNAs have been reported in various diseases and impaired
wound healing. One of the miRNAs, MiR-210 is induced in hypoxic situations and targets E2f3
resulting in reduced keratinocyte proliferation in wound healing. MiR-200b decreases
angiogenesis by steering globin transcription factor 2 and VEGFR210. Similarly various
miRNAs like miR-130a, miR-21, miR-146a, miR-198 and miR26a are involved in diabetic
wounds. They affect epithelization, delay inflammation, fibroblast migration, keratinocyte
migration, re-epithelialization and angiogenesis
13
1.8.Diabetes foot ulceration
Diabetic foot ulcers are an injury to all layers of skin, necrosis or gangrene that usually occur on
the soles of the feet, as a result of peripheral neuropathy or peripheral arterial disease in diabetes
mellitus (DM) patients.1,2 Understanding diabetic foot ulcers include necrosis or gangrene.
Diabetic gangrene is a tissue death caused by a blockage of blood vessels (ischemic necrosis)
due to micro emboli atherothrombosis which is caused by occlusive peripheral vascular disease
that accompanies diabetics as a chronic complication of diabetes itself. Diabetic foot ulcers can
be followed by bacterial invasion resulting in infection and decay, can occur in any part of the
body especially in the distal part of the lower leg(Gale and Gillespie, 2001)
1.8.1. Epidemiology of diabetic foot infection
Diabetic foot is a severe public health issue, yet rare studies investigated its global epidemiology.
Here we performed a systematic review and meta-analysis through searching PubMed,
EMBASE, ISI Web of science, and Cochrane database. We found that that global diabetic foot
ulcer prevalence was 6.3% (95%CI: 5.4–7.3%), which was higher in males (4.5%, 95%CI: 3.7–
5.2%) than in females (3.5%, 95%CI: 2.8–4.2%), and higher in type 2 diabetic patients (6.4%,
95%CI: 4.6–8.1%) than in type 1 diabetics (5.5%, 95%CI: 3.2–7.7%). North America had the
highest prevalence (13.0%, 95%CI: 10.0–15.9%), Oceania had the lowest (3.0%, 95% CI: 0.9–
5.0%), and the prevalence in Asia, Europe, and Africa were 5.5% (95%CI: 4.6–6.4%), 5.1%
(95%CI: 4.1–6.0%), and 7.2% (95%CI: 5.1–9.3%), respectively. Australia has the lowest (1.5%,
95%CI: 0.7–2.4%) and Belgium has the highest prevalence (16.6%, 95%CI: 10.7–22.4%),
followed by Canada (14.8%, 95%CI: 9.4–20.1%) and USA (13.0%, 95%CI: 8.3–17.7%). The
patients with diabetic foot ulcer were older, had a lower body mass index, longer diabetic
duration, and had more hypertension, diabetic retinopathy, and smoking history than patients
without diabetic foot ulceration(Deshpande et al., 2008).
1.8.2. Pathophysiology of Diabetic foot infection
In DM patients there is an increased occurrence of the main risk of the occurrence and
development of diabetic foot ulcers, namely peripheral neuropathy, peripheral vascular disease
and disruption of response to infection. In addition, in DM there is a wound healing disorder that
increases the risk of infection. Neuropathy in DM manifests against motor, sensory and
autonomic. Damage to the innervation of the leg muscles causes an imbalance between flexion
and leg extension, resulting in deformity and change of pressure points. Gradually, it will cause
skin damage that develops into ulcers. Autonomic neuropathy lowers the activity of oil glands
and sweat so that the foot moisture is reduced and susceptible to injury. Sensory neuropathy
lowers the pain threshold so that it is often unaware of the existence of the wound until the
wound worsens.17 In peripheral arteries, hyperglycemia causes endothelial dysfunction and
blood vessel muscle, as well as decreased vasodilator production by the endothelium resulting in
constriction. Hyperglycemia in DM increases thromboxane A2, namely vasoconstrictor and
14
aggregate platelet aggregates, resulting an increased risk of plasma hypercoagulability.
Hypertension and dyslipidemia also contribute to the occurrence of peripheral arterial disease.
The explanation above will lead to occlusive arterial disease which then causes ischemia of the
lower extremities and increases the risk of ulcers. The formed ulcers will be easily infected,
develop into gangrene and end up with a lower leg amputation (below knee amputation). In DM,
there is a decreasing in peripheral soft tissue healing ability that leads to ulcers. In diabetes,
especially in the advanced stage where the structure of skin tissue, nerves, blood vessels and
other support tissues have been damaged, so the control of blood glucose is no longer enough to
fix them. Slow wound healing in DM will increase the risk of wound complications that will
further slow wound healing. These complications include infections (including cellulitis,
abscesses and osteomyelitis), gangrene and septicemia.(Oliver and Mutluoglu, 2022)
1.8.3. Types of diabetic foot ulcers
According to Edmon diabetic foot ulcers are divided into 2 groups, namely:
 Neuropathic ulcers
Feet is warm, perfusion is still good with pulsation still palpable, perspiration is reduced, skin
dry and cracked.
 Neuroischemic ulcers
Feet is colder, not palpable pulsation, thin skin, smooth and without hair, subcutaneous tissue
atrophy, intermittent claudication and rest pain may not be present due to neuropathy.
1.8.4. Bacterial profile of Diabetic foot infection
In the present study, out of 150 PWD, 56 patients were below 50 years and 94 patients were above 50
years. In our study, 122 patients were males and 28 were females. In this study, there were 43 (28.6%)
polymicrobial cases, 96 (64%) monomicrobial cases, and in 11 (7.3%) cases, the culture was sterile.
Gram-positive bacterial growths were present in 73 (41%) cases, whereas Gram-negative growth was
seen in 106 (59%) cases. The most common single bacterial growth was that of S. aureus (27%), followed
by E. coli (20%), and Enterococcus spp. (15.7%). Among the isolates, 59/112 (53%) of the
Gram-negative bacilli were ESBL producers, 19/46 (41%) were MRSA, and 5/27 (19%) were VRE. In
the present study, most of the Enterobacteriaceae culture isolates were sensitive to amikacin (90%),
imipenem (89%), meropenem (84%), ertapenem (76%), and piperacillin-tazobactam combination (73%).
In our study, most of the Pseudomonas culture isolates were sensitive to amikacin (90%), imipenem
(72%), meropenem (70%), and piperacillin-tazobactam combination (74%). Most of the Staphylococcus
culture isolates were sensitive to linezolid (100%), daptomycin (100%), tigecycline (89%), teicoplanin
(84%), and gentamicin (83%). In our study, most of the Enterococcus culture isolates were sensitive to
linezolid (100%), daptomycin (100%), teicoplanin (89%), and tigecycline (74%) (Xie et al., 2017).
15
1.8.5. Staphylococcus aureus
Staphylococcus aureus is Gram-positive bacteria (stain purple by Gram stain) that are cocci-
shaped and tend to be arranged in clusters that are described as “grape-like.” On media, these
organisms can grow in up to 10% salt, and colonies are often golden or yellow (aureus means
golden or yellow). These organisms can grow aerobically or anaerobically (facultative) and at
temperatures between 18 C and 40 C. Typical biochemical identification tests include catalase
positive (all pathogenic Staphylococcus species), coagulase positive (to
distinguish Staphylococcus aureus from other Staphylococcus species), novobiocin sensitive (to
distinguish from Staphylococcus saprophyticus), and mannitol fermentation positive (to
distinguish from Staphylococcus epidermidis). MRSA strains carry a mec gene on the bacterial
chromosome, which is a component of the larger Staphylococcal chromosomal
cassette mec (SCCmec) region, conferring resistance to multiple antibiotics depending on the
SCCmec type. The mec gene encodes the protein PBP-2a (penicillin-binding protein 2a). PBP-2a
is a penicillin-binding protein (PBP), or essential bacterial cell wall enzyme that catalyzes the
production of the peptidoglycan in the bacterial cell wall. PBP-2A has a lower affinity to bind to
beta-lactams (and other penicillin-derived antibiotics) when compared to other PBPs, so PBP-2A
continues to catalyze the synthesis of the bacterial cell wall even in the presence of many
antibiotics. As a result, S. aureus strains that synthesize PBP-2A can grow in the presence of
many antibiotics, and these MRSA strains are resistant to many antibiotics. MRSA strains tend to
be resistant to methicillin, nafcillin, oxacillin, and cephalosporins.(Taylor and Unakal, 2022)
1.8.6. Escherichia coli
E. coli is a Gram-negative, facultative anaerobe, nonsporulating coliform bacterium. Cells are

typically rod-shaped, and are about 2.0 μm long and 0.25–1.0 μm in diameter, with a cell volume
of 0.6–0.7 μm3.Antibiotics can effectively treat E. coli infections outside the digestive tract and
most intestinal infections but are not used to treat intestinal infections by one strain of these
bacteria. The flagella which allow the bacteria to swim have a peritrichous arrangement.
Optimum growth of E. coli occurs at 37 °C (99 °F), but some laboratory strains can multiply at
temperatures up to 49 °C (120 °F).[28] E. coli grows in a variety of defined laboratory media,
such as lysogeny broth, or any medium that contains glucose, ammonium phosphate monobasic,
sodium chloride, magnesium sulfate, potassium phosphate dibasic, and water. Growth can be
driven by aerobic or anaerobic respiration, using a large variety of redox pairs, including the
oxidation of pyruvic acid, formic acid, hydrogen, and amino acids, and the reduction of
substrates such as oxygen, nitrate, fumarate, dimethyl sulfoxide, and trimethylamine N-
oxide.[29] E. coli is classified as a facultative anaerobe. It uses oxygen when it is present and
available. It can, however, continue to grow in the absence of oxygen using fermentation or
anaerobic respiration. The overall susceptibility patterns of E. coli show significantly high
resistance rates to erythromycin (89.4%), amoxicillin (86.0%) and tetracycline (72.6%) were
documented (p=0. 001). On the other hand, significantly high degree of sensitivity rates to
16
nitrofurantoin (96.4%), norfloxacin (90.6%), gentamicin (79.6%) and ciprofloxacin were
detected.
1.8.7. Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common encapsulated, Gram-negative, strict aerobic (although can

grow anaerobically in the presence of nitrate), Rod-shaped bacterium that can cause disease in
plants and animals, including humans. A species of considerable medical importance, P.
aeruginosa is a multidrug resistant pathogen recognized for its ubiquity, its intrinsically advanced
antibiotic resistance mechanisms, and its association with serious illnesses – hospital-acquired
infections such as ventilator-associated pneumonia and various sepsis syndromes. P. aeruginosa
grows well on LB broth, but can also utilize a wide range of compounds as sole carbon and/or
nitrogen sources. To study growth on these sole nutrient sources, various defined minimal media
are used to grow P. aeruginosa such as MOPS (3-(N-Morpholino) Propane-Sulfonic Acid)
medium, M9, or M63. All most all the clinical isolates of P. aeruginosa were resistant to
penicillin, ampicillin, cefixime, and cefpodoxime, 75% isolates were resistant to levofloxacin,
tobramycin, gentamicin, and ciprofloxacin, 25% were resistant to piperacillin, amikacin,
aztreonam, ceftriaxone, and cefotaxime. 75% isolates were intermediately resistant to ceftriaxone
and cefotaxime, 25% were intermediately resistant to ceftazidime. 100% isolates were sensitive
to imipenem, and meropenem, 75% isolates were sensitive to piperacillin, amikacin, aztreonam,
and ceftazidime, while 25% were sensitive to levofloxacin, tobramycin, gentamicin and
ciprofloxacin(Bhuiya et al., 2018).
1.9. Moringa oleifera
Moringa oleifera is a fast-growing, drought-resistant tree of the family Moringaceae, native to

the Indian subcontinent. Common names include moringa, drumstick tree (from the long,
slender, triangular seed-pods), horseradish tree (from the taste of the roots, which resembles
horseradish), and ben oil tree or benzolive tree. It is widely cultivated for its young seed pods
and leaves, used as vegetables and for traditional herbal medicine. It is also used for water
purification. Although listed as an invasive species in several countries. The genus name
Moringa derives from the Tamil word, murungai, meaning "twisted pod", alludes to the young
fruit. The species name oleifera is derived from the Latin words oleum "oil" and ferre "to
bear".(Makkar et al., 2007)
1.9.1. Traditional medicine and research
The bark, sap, roots, leaves, seeds and flowers are used in traditional medicine. Research has
examined how it might affect blood lipid profiles and insulin secretion. Extracts from leaves
contain various polyphenols, which are under basic research to determine their potential effects
in humans. Despite considerable preliminary research to determine if moringa components have
bioactive properties, there is no high-quality evidence to indicate that it has any effect on health
or diseases.(Muhammad et al., 2016)
17
1.9.2. Antibacterial activity of Moringa oleifera
Antibacterial activity of Moringa oleifera extracts on bacterial isolates showed that Moringa
oleifera leaf ethanol (MLE) extract had the broadest spectrum of activity on the test bacteria. The
result exhibited that MLE had antimicrobial activity against five bacterial isolates. P.
aeruginosa, P. Voulgaris, B. subtills, S epidermidis, and S mutans. Moringa oleifera is a good
source of various phytochemicals like alkaloids, flavonoids, glycosides, saponins, and tannins.
The antibacterial activity Moringa oleifera was clearly shown by the present study against
various test organisms like Escherichia Coli, Pseudomonas aeruginosa, Staphylococcus aureus,
Proteus vulgaris, Streptococcus mutans, Bacillus subtills, and Staphylococcus epidermidis.
1.9.3. Antidiabetic activity of Moringa oleifera
The incidence of diabetes is increasing in the developing world, with an increase in the number
of diabetes patients in younger age groups.1 The therapeutic management of diabetes without
any side effects remains a challenge. In response, there is a growing interest in evaluating herbal
remedies, which are seen to be less toxic and to have negligible side effects. Moringa oleifera is
indigenous to the Indian subcontinent and is widely used in Ayurvedic medicine for the
treatment of cardiac and circulation problems. Phytochemical investigations of M. oleifera have
revealed the presence of 4-(4′-o-acetyl-α-l-rhamnopyranosyloxy) benzyl isothiocyanate, 4-(-l-
rhamnopyranosyloxy) benzyl isothiocyanate, niazimicin, pterygospermin, benzyl isothiocyanate,
and 4-(α-l-rhamnopyranosyloxy) benzyl glucosinolates. Moringa is a nutritionally rich botanical
with a high polyphenol content, including phenolic acids, flavonoids, and glucosinolates (Gupta
et al., 2012). In preclinical studies, fiber content of MO leaves mediated quercetin-3-glucoside to
improve glucose tolerance. Phenolic glycosides from the fruit show anti-inflammatory effects by
inhibiting nitric oxide. Dipeptide and urea derivatives from MO roots also have anti-
inflammatory and antinociceptive effects. The ethanolic seed extract produced
immunosuppressive and anti-inflammatory effects by inhibiting leukocytes and splenocytes as
well as histamine release from mast cells(“Moringa oleifera | Memorial Sloan Kettering
Cancer Center,” n.d.)
18
Chapter 2
2.1. Material
The FTIR (Fourie transform infrared) spectra, using KBr pellets under the scanning range from
4000 – 500cm-1 with a weight ratio of 5:200mg, were measured using Shimadzu FT-IR 8400
spectrometer at room temperature. The absorption spectra of an aqueous solution of the sample
were recorded using, a quartz cell 1.0cm path length equipped, Shimadzu UV-is 650 PC
spectrophotometer. A Metrohm model 827 pH meter was used for the measurements of the pH of
the sample.
2.2. Methodology
2.2.1. Collection of Moringa Oleifera leaves
Each leaf was destalked and kept in sunlight to dry for 10 minutes and after that for complete air
drying was kept at room temperature. To prevent fungal growth continuous turning of the leaflet
was ensured for up to two weeks. Afterward, they were kept away from sunlight to keep active
compounds present in the leaf safe. Then, leaves were ground to a fine powder using a sterile
pestle and mortar up to 45g using measuring balance.
2.2.2. Preparation of ethanolic extraction
The ethanolic leaf extract was prepared by soaking 45 g of ground powder in 500ml 70%
ethanol in a sterile jar at room temperature for 72 hours. After 72 hours the moringa suspension
in ethanol was filtered through Whatman® qualitative filter paper, ashless Grade- 41. circles,
diam. 90mm, pore size 20µm. The filtrate was collected and stored in a sterile capped flask at
room temperature for future process.
2.2.3. Media preparation
By following the standard protocols recommended by manufacturers, washed flasks were

collected and then placed in a hot air oven at 170 0 C for 90 min. The weight in grams of
powdered media was calculated according to the directed recipes on the media container and
then powdered was weighed using measuring balance and poured into the sterile flask containing
distilled water in the required volume. Afterward, the flask was kept for autoclave under 1.5 psi
pressure at 1210 C for 15 minutes. Following Media were prepared for the processing of the
sample
 Brain Heart Infusion (BHI) was used for the enrichment of Microbes present in
transport media
 Nutrient Agar (NA) was used for mixed growth
19
 Eosin Methylene Blue (EMB) for the selective culturing of Enterobacteriaceae
spp.
 Pseudomonas Cetrimide Agar (PCA) for selective culturing of Pseudomonas
aeruginosa
 Mannitol Salt Agar (MSA) for the growth of Staphylococcus Aureus
 Triple Sugar Iron agar (TSI) for identification of Enterobacter’s
 MacConkey agar, differential agar for lactose fermenter and non-fermenter
2.2.4. Preparation of Swabs for sampling
Sterile disposable cotton swabs were collected along with commercial sterile 0.9% saline
solution were purchased. Under the sterile condition, near the flame about 1ml sterile saline was
transferred into each cotton swab using autoclaved tips and a pipette. Afterward, autoclaved
paper tapes
2.2.5. Collection of Samples
Swab samples were collected from the infected wounds of diabetic patients from the Nishtar
Medical University and Hospital, Multan. The samples were collected among the patients in the
age group of 25 to 60 years. The swabbing was done from muscles, pus, and bones from the
wound for sample collection using sterile cotton swabs dipped in saline, to maintain the viability
of sample microbes, as a transport media.
2.2.6. Sample Enrichment
Brain heart infusion was used for the enrichment of sample microbes. 10ml of autoclaved BHI
broth was poured into sterile labeled falcon tubes under sterile conditions. Then swabs were
dipped into the falcon tube containing the sample additionally the saline (transport media) was
also poured into the same falcon tube. The falcon tubes along with the control, falcon tube
containing only BHI broth, were then placed in the shaking incubator at 37 0C overnight.
2.2.7. Subculturing
After enrichment culturing, BHI broth got turbid which showed bacterial growth. Then,
autoclaved molten agar of MSA, PCA, EMB, and MacConkey agar was poured into separate
sterile Petri-plates in the volume of 11-15 ml near the flame. After molten agar in Petri-plates got
cooled and turned into solid agar, swabs dipped in the turbid BHI broth were used to spread the
enriched sample on all the agar plates for the isolation of different bacterial species.
20
2.2.8. Identification and conformation of bacterial species
For further confirmation of Bacterial species Triple sugar Iron slants were prepared and
incubated at 370C FOR 24 hours after inoculated with bacterial colonies differentiated through
MacConkey Agar. Change in color of butt and slops along with presence of black line and sign
of gas production were noted after the incubation of slants. Meanwhile catalase test, oxidase test,
coagulase test and gram staining were done for the complete identification of bacterial species.
2.2.9. Kirby Bauer Disc Diffusion Test:
 Disc diffusion method for bacterial antibiotic susceptibility profile
Bacterial suspension was prepared by adding some bacterial colonies in 3 ml of saline.

Optical density of bacterial suspension at λ of 420 nm were taken using a quartz cell 1.0cm
path length equipped, Shimadzu UV-is 650 PC spectrophotometer. After calculating the
optical density of bacterial suspension optimized diluted bacterial suspension were formed
using 10-fold serial dilution. Meanwhile, autoclaved Mueller Hinton Agar were poured into
the petri plates. After plating the bacterial sample in volume of 100 µl were transferred into
petri plates and by using sterile cotton swabs bacterial suspension got spread all over the
plate. A set of antibiotic disks, which includes Clindamycin, Vancomycin, Ampicillin,
Ciprofloxacin, Erythromycin, and, Oxalic Acid were placed on the agar plate using sterile
forceps near the flame followed by the incubation period of 24 hours at 37 0C. After the
incubation Zone of inhibition were calculated using Vernier caliper in unit of mm.
 Agar well diffusion test for the antibiotic activity of Moringa Oleifera
Bacterial suspension was prepared by adding some bacterial colonies in 3 ml of saline.

Optical density of bacterial suspension at λ of 420 nm were taken using a quartz cell 1.0cm
path length equipped, Shimadzu UV-is 650 PC spectrophotometer. After calculating the
optical density of bacterial suspension optimized diluted bacterial suspension were formed
using 10-fold serial dilution. Meanwhile, autoclaved Mueller Hinton Agar were poured into
the petri plates. After plating the bacterial sample in volume of 100 µl were transferred into
petri plates and by using sterile cotton swabs bacterial suspension got spread all over the
plate. For the preparation of well a 1ml sterile tips were used to bore with and sterile forceps
was used to pick the extra agar. After that 100 µl of moringa extract with the concentration of
90mg/100µl were transferred into the well using sterile pipette. While ciprofloxacin was used
as a positive control. After the incubation period of 24 hours and 37 0C the zone of inhibition
was calculated and compared with the zone of positive control.
21
2.2.10. MIC and MBC assay
Minimal Inhibitory Concentration and Minimal Bactericidal Concentration of moringa extract

was determined against all the bacterial species isolated using 96 well flat bottom micro titter
plates (Polystyrene, 96 TM BD, USA). 100µl of sterile saline were transferred in each well.
After that 2-fold serial dilution of moringa extract were done by adding extract with the
concentration of 90mg/100µl in volume of 100µl in well no.1 and diluted the extract with 2 folds
up to well no.10, while keep the leaving wells 11 and 12 for positive and negative controls. After
serial dilution, 100µl of bacterial suspension in concentration of 10 5 CFU/ ml were added in each
well from 1 -10 and in positive control will extract were added in negative control followed by
the incubation of 24 hours at 370C. After incubation results were noted and for further
confirmation aliquots of 10µl were added to petri plates for spreading from the well of MIC and
MBC.
22
Chapter no. 3
Results
3.1. Immunologic History of Patients with DFIs
Table 1: Immunologic History of Patients with DFIs
Total number of patients 30

Male (%age) 24(82)
Female (%age) 6(18)
Amputation Rate 84 %
Age (years) 32-63
Literacy rate 5%
Diabetes (type I / type II) 9/21
Duration of Diabetes 3-10 years
Ulcer site Right foot sole (83 %)
Ulcer size 4-9 cm2
Hypertension rate 69 %
Smoking rate 77 %
Major complications Nephropathy
Neuropathy
Charcot foot
Loss of sensation
Skin infection
Amputation
Patient history showing 82% males &18% females having amputation rate 84% with major
complications involving neuropath, skin infection, amputation etc. Duration of diabetes, site &
size of ulcer has been shown.
23
3.2. Bacterial Prevalence
Table 2: Prevalence of Pathogenic Isolates from Diabetic Foot Infections

Total no. of samples Total number of Name of Bacteria % Age
isolates
Staphylococcus 100 %
Aureus
Pseudomonas 100%
Aeruginosa
Salmonella 80%
30 108 Typhimurium
Escherichia 40%
coli
Klebsiella 20%
pneumoniae
Proteus 20%
mirabilis
Prevalence of 6 bacterial strains from diabetic foot infections.
3.3. Identification of Bacteria

3.3.1. Bacterial identification through culture media
By morphology colors of colonies following 6 bacteria were isolated
 Staphylococcus Aureus shows typical yellow colonies on MSA

 Pseudomonas Aeruginosa shows yellow colonies on Pseudomonas cetrimide agar
 Salmonella Typhimurium shows pink colonies on EMB agar
 Escherichia coli shows metallic green colonies on EMB agar
 Klebsiella pneumoniae purple colonies on EMB agar
 Proteus mirabilis white colonies on MacConkey Agar
24
3.3.2 Biochemical testing for Bacterial identification
Table 3. Biochemical test of Bacteria
Bacterial Specie Coagulase Oxidase Catalase TSI slants result

test test test
Staphylococcus Positive Negative Positive Y: butt/ Y: slop
Aureus H2S production: -
ve
Gas production: -
ve
Pseudomonas Negative Positive Positive R: butt/ R: slop

Aeruginosa H2S production: -
ve
Gas production: -
ve
Salmonella Positive Negative Positive R: butt/ R: slop

Typhimurium H2S production:
+ve
Gas production: -
ve
Escherichia Negative Negative Positive Y: butt/ Y: slop

coli H2S production: -
ve
Gas production:
+ve
Klebsiella Negative Negative Positive R: butt/ Y: slop

pneumoniae H2S production:
+ve
Gas production: -
ve
Proteus Negative Negative Positive R: butt/ Y: slop

mirabilis H2S production: -
ve
Gas production: -
ve
Biochemical tests of 6 bacterial species which were isolated from diabetic foot infection
25
Figure 1: Growth of Staphylococcus Aureus on MSA
MSA MSA
Typical yellow colonies of staphylococcus aureus are shown on MSA agar.
Figure 2: Growth of Salmonella Typhimurium on EMB agar
EMB EMB
Pink colonies of Salmonella Typhimurium are shown on EMB agar.
Figure 3: Growth of Pseudomonas Aeruginosa on PCA
PCA PCA
Typical yellow colonies of Pseudomonas aeruginosa are shown on PCA.
26
Figure 4: Growth of Escherichia coli on EMB agar
EMB EMB
Metallic green colonies of Escherichia coli are shown on EMB
Figure 5: Growth of Klebsiella pneumoniae on EMB agar
EMB EMB
Purple colonies of Klebsiella pneumoniae are shown on EMB
Figure 6: Growth of Proteus mirabilis on MacConkey agar
MAC
MAC
White colonies of Proteus mirabilis are shown on EMB
27
3.4. Determination of Antibiotic Resistance profile of Isolated bacterial species
by Kirby Bauer Disk diffusion Test
Table 4: Antimicrobial Sensitivity Potential of different Antibiotics against Staphylococcus
aureus
Name of
Bacterial sp. Serial Antibiotics Disc Susceptible Resistant (%Age)
No. used (conc. µg) (%Age)
1 Ciprofloxacin (5) 78 21
2 Erythromycin (15) 15 84
Staphylococcus
Aureus 3 Fuscidic Acid (10) 10 89
4 Oxalic Acid (1) 0 100
5 Clindamycin (2) 21 78
Antimicrobial Activity of Staphylococcus Aureus was determined against different antimicrobial

groups including Quinolones, Macrolides, Lincosamides and Miscellaneous antimicrobials. Most
susceptible antibiotics was Ciprofloxacin and the resistant antibiotic was Fuscidc and Oxalic
Acid
28
Graph No. 1: Antimicrobial Sensitivity Potential of different Antibiotics
against Staphylococcus Aureus (n=30)
120
100
100
89
84
78 78
80
percentage
60
40
21 21
20 16
10
0
0
Ciprofloxacin (5) Erythromcin (15) Fuscidic Acid(10) Oxalic Acid(1) Clindamycin (2)
Antibiotics (conc. µg )
Susceptible (%age) Resistant (%age)
Graphical representation of antimicrobial potential of 4 different antibiotics is shown against
Staphylococcus Aureus
Figure 7: Antimicrobial Sensitivity Potential of different Antibiotic against

Staphylococcus Aureus (n=30)
Staph Staph
Mueller Hinton agar plate showing zone of Inhibition of different group of antibiotics against
29
Table 5: Antimicrobial Sensitivity Potential of different Antibiotics against
Pseudomonas aeruginosa
Name of
1 Linezolid (30) 0 100
2 Colistin (10) 10 89
Pseudomonas
Aeruginosa 3 Doripenem (10) 0 100
5 Ticarcillin (2) 0 100
Antimicrobial Activity of Pseudomonas aeruginosa was determined against different

antimicrobial groups including Carbapenems, Penicillin, Macrolides, Lincosamides and
Miscellaneous antimicrobials. Most susceptible antibiotics was Clindamycin and the resistant
antibiotic were Linezolid, Doripenem, and Ticarcillin.
30
against Pseudomonas aeruginosa (n=30)
120
100 100
100
89
78 78
80
Percentage
60
40
21
20
10
0 0 0
0
linezolid (30) Colistin (10) Doripenem (10) Clindamycin (2) tricarcillin (75)

Pseudomonas Aeruginosa (n=30)
Pseudo Pseudo
Pseudomonas aeruginosa
31
Table6: Antimicrobial Sensitivity Potential of different Antibiotics against
Salmonella typhimurium Isolates (n=24)
Name of
1 Streptomycin (10) 0 100
2 Vancomycin (15) 10 89
Salmonella
typhimurium 3 Clindamycin (2) 10 89
5 Ampicillin (20) 0 100
Antimicrobial Activity of Salmonella typhimurium was determined against different

Miscellaneous antimicrobials. Most susceptible antibiotics were Clindamycin and Vancomycin
and the resistant antibiotic were Streptomycin, Ampicillin, and linezolid
32
against Salmonella Typhimurium (n=24)
120
100 100
100 94
89 89
Percentage
80
60
40
20
10 10
5
0 0
0
Streptomycin (10) vancomycin (30) Clindamycin (2) Linezolid (30) Ampicillin (20)

Salmonella typhimurium (n=24)
Salmonella Salmonella
Salmonella typhimurium
33
Table 7: Antimicrobial Sensitivity Potential of different Antibiotics
Escherichia coli
Name of
Escherichia
coli 3 Clindamycin (2) 25 75
Antimicrobial Activity of Salmonella typhimurium was determined against different

Miscellaneous antimicrobials. Most susceptible antibiotics was Clindamycin and the resistant
antibiotic were Streptomycin, and Ampicillin
34
against Escherichia coli (n=12)
120
100
100 94
80 76 75
percentage
60
40
25
21
20 16
5
0 0
0
sStreptomycin (10) Vancomycin (15) Clindamycin (2) Linzolid (30) Ampicillin (20)
Escherichia coli

Escherichia coli (n=24)
E. coli E. coli
Escherichia coli
35
Table 8: Antimicrobial Sensitivity Potential of different Antibiotics against Klebsiella
pneumoniae
Name of
2 Colistin (10) 33 49
Klebsiella
pneumoniae 3 Doripenem (10) 0 100
5 Ticarcillin (2) 16 84
Antimicrobial Activity of Klebsiella pneumoniae was determined against different antimicrobial

groups including Carbapenems, Penicillin, Macrolides, Lincosamides and Miscellaneous
antimicrobials. Most susceptible antibiotics was Clindamycin and the resistant antibiotic were
Linezolid and Doripenem.
36
against Klebsiella pneumoniae (n=6)
120
100 100
100
78
80
66
Axis Title
60
49
44
40 33
20 16
0 0
0
Linezolid (30) Colistin (10) Doripenem (10) Clindamycin (1) Ticarcilim (2)
Klebsiella pneumoniae

Klebsiella pneumoniae (n=6)
Kleb Kleb
37
Table 9: Antimicrobial Sensitivity Potential of different Antibiotics against Proteus mirabilis
Name of
Proteus
mirabilis 3 Clindamycin (2) 50 34
Antimicrobial Activity of Proteus mirabilis was determined against different antimicrobial

groups including Carbapenems, Penicillin, Macrolides, Lincosamides and Miscellaneous
antimicrobials. Most susceptible antibiotics was Clindamycin and the resistant antibiotic were
Streptomycin, linezolid, and Ampicillin
38
against Proteus mirabilis (n=6)
120
100 100 100

100
Percentage
80
60
49 50
40 33 34
20
0 0 0
0
streptomycin (10) Vancomycin (15) Clindamycin (2) Linwzolid (30) Ampicilin (20)
Proteus mirabilis

Proteus mirabilis (n=6)
Proteus Proteus
Proteus mirabilis
39
3.5. Determination of antimicrobial activity of ethanolic extract of Moringa
oleifera by Agar well diffusion Method
Table 10: Antimicrobial potential of ethanolic extract of Moringa oleifera

against Staphylococcus aureus by agar well method (n=30)
Name of Susceptible Resistant

Name of Isolate Serial No. antimicrobial (%age) (%age)
agent used
1 Oxalic acid
(1µg) 21 78
Staphylococcus
Aureus
Ethanolic extract
2 Of moringa
(90µg) 100 0
Determination of antimicrobial potential of ethanolic leaf extract of Moringa oleifera against

Staphylococcus aureus using ciprofloxacin as a standard
40
Graph no. 7: Antimicrobial potential of ethanolic extract of Moringa oleifera
against Staphylococcus aureus by agar well method (n=30)
120
100
100
78
80
percentage
60
40
21
20
0
0
Oxalic acid (1µg) ethanolic leaf extract (90µg)
Antimicrobial agent (conc.)
Graphical representation of antimicrobial potential of Moringa oleifera extract against

Staphylococcus aureus by agar Well diffusion Method (n=30)
Figure 13: Antimicrobial Activity of Moringa oleifera extract against

Staphylococcus aureus
41
against Pseudomonas Aeruginosa by agar well method (n=30)

agent used
1
Linezolid (30µg) 0 100
Pseudomonas
Aeruginosa
Ethanolic extract
2 Of moringa
(90µg) 80 20

Pseudomonas Aeruginosa using ciprofloxacin as a standard
42
against Pseudomonas Aeruginosa by agar well method (n=30)
120
100
100
80
80
percentage
60
40
20
20
0
0
Linzolid (30µg) ethanolic leaf extract (90µg)

Pseudomonas Aeruginosa by agar Well diffusion Method (n=30)

Pseudomonas Aeruginosa
43
against Salmonella typhimurium by agar well method (n=24)

agent used
1 Ampicillin
(20µg) 0 100
Salmonella
typhimurium
Ethanolic extract
2 Of moringa
(90µg) 69.6 29.1
29.1

Salmonella typhimurium using ciprofloxacin as a standard
44
against Salmonella typhimurium by agar well method (n=24)
120
100 100
100
80
percentage
60
40
29.1
20
0
0
Ampicillin (20µg) ethanolic leaf extract (90µg)
Graphical representation of antimicrobial potential of Moringa oleifera moringa extract

against Salmonella typhimurium by agar Well diffusion Method (n=24)
Figure 15: Antimicrobial Activity of Moringa oleifera extract against Salmonella

typhimurium
45
against Escherichia coli by agar well method (n=12)

agent used
1 Streptomycin
(10µg) 0 100
Escherichia
coli
Ethanolic extract
2 Of moringa
(90µg) 91.66 8.44

Escherichia coli using ciprofloxacin as a standard
46
against Escherichia coli by agar well method (n=12)
120
100
100 91.66
80
percentage
60
40
20
8.44
0
0
Streptomycin (10µg) ethanolic leaf extract (90µg)

Escherichia coli by agar Well diffusion Method (n=12)
Figure 16: Antimicrobial Activity of Moringa oleifera extract against Escherichia

coli
47
against Klebsiella pneumoniae s by agar well method (n=6)

agent used
1 Doripenem (10
µg) 0 100
Klebsiella
pneumoniae
Ethanolic extract
2 Of moringa
(90µg) 66.66 33.44

Klebsiella pneumoniae using ciprofloxacin as a standard
48
against klebsiella pneumoniae by agar well method (n=6)
120
100
100
80
66.66
percentage
60
40 33.44
20
0
0
Doripenem (10µg) ethanolic leaf extract (90µg)
Graphical representation of antimicrobial potential of Moringa oleifera extract

against klebsiella pneumoniae by agar Well diffusion Method (n=6)

49
against Proteus mirabilis by agar well method (n=6)

agent used
1 Ampicillin
(20µg) 0 100
Proteus
mirabilis
Ethanolic extract
2 Of moringa
(90µg) 100 0

proteus mirabilis using ciprofloxacin as a standard
50
against Proteus mirabilis by agar well method (n=6)
120
100 100
100
80
percentage
60
40
20
0 0
0
Ampicillin (20µg) ethanolic leaf extract (20µg)

Proteus mirabilis by agar Well diffusion Method (n=6)
Figure 18: Antimicrobial Activity of Moringa oleifera extract against Proteus

mirabilis
51
Table 16: determination of MIC and MBV values of Ethanolic extract of
Moringa Oleifera against Staphylococcus aureus Strains
Name of Name of Isolate Isolate MIC (conc. µg) MBC (conc. µg)
compound no.
1 1.406 µg/ 100µl 2.812µg/ 100µl
2 1.406µg/ 100µl 2.812µg/ 100µl
3 5.625µg/ 100µl 11.25µg/ 100µl
4 2.821µg/ 100µl 5.625µg/ 100µl
5 2.821µg/ 100µl 5.625µg/ 100µl
6 5.625µg/ 100µl 11.25µg/ 100µl
7 11.25µg/ 100µl 22.5µg/ 100µl
8 2.821µg/ 100µl 5.625µg/ 100µl
9 5.625µg/ 100µl 11.25µg/ 100µl
10 5.625µg/ 100µl 11.25µg/ 100µl
11 1.406 µg/ 100µl 2.812µg/ 100µl
12 5.625µg/ 100µl 11.25µg/ 100µl
13 1.406 µg/ 100µl 2.812µg/ 100µl
14 2.821µg/ 100µl 5.625µg/ 100µl
Ethanolic extract Staphylococcus 15 11.25µg/ 100µl 22.5µg/ 100µl
of Moringa aureus 16 1.406 µg/ 100µl 2.812µg/ 100µl
Oleifera 17 2.821µg/ 100µl 5.625µg/ 100µl
18 5.625µg/ 100µl 11.25µg/ 100µl
19 11.25µg/ 100µl 22.5µg/ 100µl
20 1.406 µg/ 100µl 2.812µg/ 100µl
21 1.406 µg/ 100µl 2.812µg/ 100µl
22 2.821µg/ 100µl 5.625µg/ 100µl
23 11.25µg/ 100µl 22.5µg/ 100µl
24 1.406 µg/ 100µl 2.812µg/ 100µl
25 2.821µg/ 100µl 5.625µg/ 100µl
26 1.406 µg/ 100µl 2.812µg/ 100µl
27 1.406 µg/ 100µl 2.812µg/ 100µl
28 2.821µg/ 100µl 5.625µg/ 100µl
29 5.625µg/ 100µl 11.25µg/ 100µl
30 11.25µg/ 100µl 22.5µg/ 100µl
Determination of MIC and MBV values of Ethanolic extract of Moringa Oleifera against
Staphylococcus aureus Strains. The average MIC value was 5.1505µg/100µl and the average
MBC value was 10.301µg/100µl.
52
Moringa Oleifera against Pseudomonas aeruginosa Strains
compound no.
1 2.8128µg/ 100µl 1.4062µg/ 100µl
2 5.625µg/ 100µl 2.812µg/ 100µl
3 2.8128µg/ 100µl 1.4062µg/ 100µl
4 5.625µg/ 100µl 2.812µg/ 100µl
5 2.8128µg/ 100µl 1.4062µg/ 100µl
6 2.8128µg/ 100µl 1.4062µg/ 100µl
7 5.625µg/ 100µl 2.812µg/ 100µl
8 5.625µg/ 100µl 2.812µg/ 100µl
9 2.8128µg/ 100µl 1.4062µg/ 100µl
10 5.625µg/ 100µl 2.812µg/ 100µl
11 11.25µg/ 100µl 5.625µg/ 100µl
12 11.25µg/ 100µl 5.625µg/ 100µl
13 11.25µg/ 100µl 5.625µg/ 100µl
14 11.25µg/ 100µl 5.625µg/ 100µl
Ethanolic extract Pseudomonas 15 11.25µg/ 100µl 5.625µg/ 100µl
of Moringa aeruginosa 16 11.25µg/ 100µl 5.625µg/ 100µl
18 11.25µg/ 100µl 5.625µg/ 100µl
19 2.8128µg/ 100µl 1.4062µg/ 100µl
20 11.25µg/ 100µl 5.625µg/ 100µl
21 11.25µg/ 100µl 5.625µg/ 100µl
22 11.25µg/ 100µl 5.625µg/ 100µl
23 2.8128µg/ 100µl 1.4062µg/ 100µl
24 11.25µg/ 100µl 5.625µg/ 100µl
25 2.8128µg/ 100µl 1.4062µg/ 100µl
26 2.8128µg/ 100µl 1.4062µg/ 100µl
27 11.25µg/ 100µl 5.625µg/ 100µl
28 11.25µg/ 100µl 5.625µg/ 100µl
29 11.25µg/ 100µl 5.625µg/ 100µl
30 11.25µg/ 100µl 5.625µg/ 100µl
Pseudomonas aeruginosa Strains. The average MIC value was 6.5623µg/100µl and the
average MBC value was 13.1246µg/100µl.
53
Moringa Oleifera against Salmonella typhimurium Strains
Name of Name of Isolate Isolate MIC (conc. µg) MBC (conc.

compound no. µg)
1 22.5µg/ 100µl 11.25µg/ 100µl
2 11.25µg/ 100µl 5.625µg/ 100µl
3 22.5µg/ 100µl 11.25µg/ 100µl
4 45µg/ 100µl 90µg/ 100µl
5 11.25µg/ 100µl 5.625µg/ 100µl
6 5.625µg/ 100µl 2.812µg/ 100µl
7 22.5µg/ 100µl 11.25µg/ 100µl
8 5.625µg/ 100µl 2.812µg/ 100µl
9 11.25µg/ 100µl 5.625µg/ 100µl
Ethanolic extract Salmonella 10 22.5µg/ 100µl 11.25µg/ 100µl
of Moringa typhimurium
11 11.25µg/ 100µl 5.625µg/ 100µl
Oleifera
12 5.625µg/ 100µl 2.812µg/ 100µl
13 22.5µg/ 100µl 11.25µg/ 100µl
14 5.625µg/ 100µl 2.812µg/ 100µl
15 11.25µg/ 100µl 5.625µg/ 100µl
16 22.5µg/ 100µl 11.25µg/ 100µl
17 5.625µg/ 100µl 2.812µg/ 100µl
18 11.25µg/ 100µl 5.625µg/ 100µl
19 11.25µg/ 100µl 5.625µg/ 100µl
20 5.625µg/ 100µl 2.812µg/ 100µl
21 11.25µg/ 100µl 5.625µg/ 100µl
22 45µg/ 100µl 90µg/ 100µl
23 11.25µg/ 100µl 5.625µg/ 100µl
24 22.5µg/ 100µl 11.25µg/ 100µl
Salmonella typhimurium Strains. The average MIC value was 21.0937µg/100µl and the
average MBC value was 42.1875µg/100µl.
54
Moringa Oleifera against Escherichia coli Strains
compound no.
1 11.25µg/ 100µl 5.625µg/ 100µl
2 5.625µg/ 100µl 2.812µg/ 100µl
3 11.25µg/ 100µl 5.625µg/ 100µl
4 5.625µg/ 100µl 2.812µg/ 100µl
Ethanolic extract Escherichia coli 5 11.25µg/ 100µl 5.625µg/ 100µl
of Moringa 6 5.625µg/ 100µl 2.812µg/ 100µl
8 11.25µg/ 100µl 5.625µg/ 100µl
9 5.625µg/ 100µl 2.812µg/ 100µl
10 5.625µg/ 100µl 2.812µg/ 100µl
11 2.821µg/ 100µl 1.406µg/ 100µl
12 11.25µg/ 100µl 5.625µg/ 100µl
Escherichia coli Strains. The average MIC value was 6.5653µg/100µl and the average MBC
value was 13.1306µg/100µl
55
Moringa Oleifera against Klebsiella Pneumonia Strains
compound no.
1 11.25µg/ 100µl 5.625µg/ 100µl
Ethanolic extract Klebsiella 2 22.5µg/ 100µl 45µg/ 100µl
of Moringa Pneumonia 3 22.5µg/ 100µl 45µg/ 100µl
5 2.812µg/ 100µl 1.406µg/ 100µl
6 11.25µg/ 100µl 5.625µg/ 100µl
Klebsiella Pneumonia Strains. The average MIC value was 12.1904µg/100µl and the average
MBC value was 24.3808µg/100µl
56
Moringa Oleifera against Proteus mirabilis Strains
compound no.
1 5.625µg/ 100µl 11.25µg/ 100µl
Ethanolic Proteus 2 2.812µg/ 100µl 5.625µg/ 100µl
extract of mirabilis 3 5.625µg/ 100µl 11.25µg/ 100µl
Moringa 4 5.625µg/ 100µl 11.25µg/ 100µl
6 5.625µg/ 100µl 11.25µg/ 100µl
Proteus mirabilis Strains. The average MIC value was 4.2185µg/100µl and the average MBC
value was 8.427µg/100µl
57
Chapter no. 4
4.1. Discussion:
Diabetic foot infections are more prevalent in patients suffering from diabetes. In our research
study, total 30 samples were collected from the depth of diabetic foot infections. There ratio of
male to female patient was 4 1, which means males were more exposed to diabetic foot
infections than females. Patients were mainly in the age group of 32-63 years. In a study
investigating the microbiology of diabetic foot infections in, Southwest Ethiopia, following
conclusions were made that correlate with our findings too. It was reported that out of 194
samples, 119 were males & 75 were females which represent prevalence of diabetic foot
infections more in males. It was reported by this study that age range of patients was between 32-
92 years (Abdissa et al., 2020). Other studies also reported that males are more susceptible to
diabetic foot infections than females (Cho et al., 2018).
The major pathogens isolated in our study were Staphylococcus aureus, Pseudomonas
aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumonia, & Proteus
mirabilis. In other studies, major pathogens isolated from Diabetic foot infections were S.aureus,
Beta-hemolytic Streptococci & Bacilli (Xie et al., 2017).Another study also reported prevalence
of P. aeruginosa and E colt along with MRSA strains of S aureus in DFIs(Markakis et al.,
2016).
The antibiotic resistance pattern of our isolated pathogens reported in our study was as followed:
(100%) & (89%) isolates of S. aureus were resistant to Oxalic Acid and Fuscidic acid
respectively while (78%) were sensitive to ciprofloxacin and (21%) to Clindamycin (100%).
Isolates of P. aeruginosa showed resistance against Doripenem, Ticarcillin and Linezolid While
some strains showed sensitivity to Colistin (10%) and Clindamycin. S. typhimurium isolates were
resistant to Ampicillin (100%), Linezolid (94%) & Streptomycin (100%) respectively. While
sensitivity was shown against Vancomycin (10%) and Clindamycin (10%) by S typhimurium
isolates. Strains of E coli shown (100%) resistance against Ampicillin and Streptomycin, while
they were (16%) & (25%) sensitive to Vancomycin and Clindamycin respectively.in case of
Klebsiella pneumoniae (100%) strains were resistant to Ampicillin and Streptomycin while,
(33%) & (66%) of K. pneumoniae strains were susceptible to Colistin and Clindamycin. All
(100%) strains of Proteus mirabilis shown resistance for Streptomycin, Linezolid, and
Ampicillin and among them (50%) & (49%) were sensitive for Clindamycin and Vancomycin.
In other study, it was reported that S. aureus isolates were sensitive to Vancomycin and
Linezolid, while resistance was shown against erythromycin, tetracycline and Gentamycin.
Another study investigating antibiotic resistance pattern of P. aeruginosa reported that these
isolates were resistant to Ticarcillin, amikacin, imipenem and tetracycline ese. While P.
aeruginosa isolates were sensitive to cefotaxime. E. coli culture isolates were sensitive to
linezolid (100%), daptomycin (100%), teicoplanin (89%), and tigecycline (74%). K. pneumoniae
58
culture isolates showed the following sensitivity pattern - amikacin (90%), imipenem (72%),
meropenem (70%), and piperacillin-tazobactam (74%). P. mirabilis culture isolates, organisms
were sensitive to amikacin (90%), imipenem (89%), meropenem (84%), ertapenem (76%), and
piperacillin-tazobactam (73%)(Jain and Barman, 2017).
In present state antibacterial potential of ethanolic leaf extract of Moringa oleifera was
determined by Disc Diffusion Method and Age Well Diffusion Method, MIC and MBC were
also determined by using 96 Well Titer Plates to counter multidrug resistant isolates of
Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli,
Klebsiella pneumonia, & Proteus mirabilis. Results of agar well diffusion method showed that S.
aureus (100%) was highly susceptible to ethanolic leaf extract of Moringa oleifera as compared
to P. aeruginosa (80%), S. typhimurium (69.9%), E. coli (91.66%), k. pneumoniae (66.66%), and
P. mirabilis (100%). It was concluded that ethanolic leaf extract of Moringa oleifera has good
antibacterial potential against clinical isolates. Different researchers worked on antimicrobial
potential of ethanolic leaf extract of Moringa oleifera (Atef et al., 2019).
The average MIC values of ethanolic leaf extract of Moringa oleifera reported in our study
against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia
coli, Klebsiella pneumonia, & Proteus mirabilis was 5.1505μg/100μl, 6.5623μg/100μl,
42.1875μg/100μl, 6.5653μg/μl, 12.1904μg/100μl, and 4.2185μg/μl respectively. Average MBC
value of ethanolic leaf extract of Moringa oleifera against Staphylococcus aureus, Pseudomonas
aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella pneumonia, & Proteus
mirabilis was 10.301μg/100μl, 13.1246μg/100μl, 21.0937μg/100μl, 13.1306μg/μl,
24.3808μg/100μl, and 8.427μg/μl respectively. In another study investigating antimicrobial
potential of GOGA against S. aureus, it was reported that at concentration of 50500ug/ml.
ethanolic leaf extract of Moringa oleifera showed maximum antibacterial activity that correlated
with our investigations.
By Disc Diffusion Method and using Scanning Electron Microscopy, it was reported that
ethanolic leaf extract of c showed greater antimicrobial activity against MRSA strains
especially(Muhammad et al., 2016) .Hence it was found that Moringa oleifera possesses good
antibacterial activity against diabetic foot infection isolates
59
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Evaluation of Antibacterial potential of Moringa oleifera

Against the isolates of Diabetic foot Infection along with the

In partial fulfillment of requirements for the degree

Institute Of Pure and Applied Biology

Bahauddin Zakariya University, Multan.

Research Supervisor ___________________

External Examiner ___________________

Research student ___________________

Viva Held on ___________________

EFFECT OF GRAPHENE OXIDE AND GALLIC ACID COMPLEX ON

Prepared by Hamdan Aali under my guidance is partial fulfillment of the

I would like to extend my appreciation to the Division of microbiology, IPAB.

The initial source of inspiration that brought me to position where I stand

1 Immunologic History of Patients with DFIs 23

1 Antimicrobial Sensitivity Potential of different 29

1 Growth of Staphylococcus aureus on MSA 26

2 Growth of Salmonella typhimurium on EMB agar 26

3 Growth of pseudomonas aeruginosa on PCA 26

4 Growth of Escherichia coli on EMB agar 27

5 Growth of Klebsiella pneumoniae on EMB agar 27

6 Growth of Proteus mirabilis on MacConkey agar 27

7 Antimicrobial Sensitivity Potential of different 29

The finding shows that Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella

1.2. Classification of Diabetes Mellitus

Primary diabetes is a group of heterogenous disorders where hyperglycemia is a common

1.3. Epidemiology of Diabetes Mellitus

1.3.1. Prevalence of Diabetes Mellitus type 1

1.3.2. Prevalence of Diabetes Mellitus type 2

1.4.Insulin Resistance and hyperglycemia

1.5.Epidemiology and Pathogenesis of complications from diabetes

1.5.1. Common mechanisms for microvascular and macrovascular diseases in diabetes

1.6.Hyperglycemia and susceptibility to Infection

1.6.1. Impairment of Cytokine Production

1.6.2. Leukocyte Recruitment Inhibition

1.6.4. Neutrophil Dysfunction

1.6.5. Macrophage Dysfunction

1.6.6. Natural Killer Cell Dysfunction

1.6.7. Inhibition of Antibodies and Complement Effector

1.7.1. Factors involve in slow healing at cellular level

1.7.2. Metabolic deficiencies as a factor for stalled healing

1.7.3. Altered miRNAs level contributes in slow healing

1.8.1. Epidemiology of diabetic foot infection

1.8.2. Pathophysiology of Diabetic foot infection

1.8.3. Types of diabetic foot ulcers

1.8.4. Bacterial profile of Diabetic foot infection

1.8.6. Escherichia coli

E. coli is a Gram-negative, facultative anaerobe, nonsporulating coliform bacterium. Cells are

1.8.7. Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common encapsulated, Gram-negative, strict aerobic (although can

1.9. Moringa oleifera

Moringa oleifera is a fast-growing, drought-resistant tree of the family Moringaceae, native to

1.9.1. Traditional medicine and research

1.9.3. Antidiabetic activity of Moringa oleifera

2.2.2. Preparation of ethanolic extraction

2.2.3. Media preparation

By following the standard protocols recommended by manufacturers, washed flasks were

2.2.4. Preparation of Swabs for sampling

2.2.5. Collection of Samples

2.2.6. Sample Enrichment

2.2.9. Kirby Bauer Disc Diffusion Test:

 Disc diffusion method for bacterial antibiotic susceptibility profile

Bacterial suspension was prepared by adding some bacterial colonies in 3 ml of saline.

Bacterial suspension was prepared by adding some bacterial colonies in 3 ml of saline.