ABSTRACT
The incidence of diabetes mellitus is increasing globally and there is a concomitant increase in the prevalence of infections associated with it. Diabetic ulcers have become a major escalating public health issue that has its morbidities, impairments and debilitating consequences. The present study was undertaken to evaluate the bacteriological and multidrug resistant profile of chronic diabetic foot ulcers in patients in Mbano metropolis. One hundred and fifty samples were collected. Samples were processed by standard microbiological methods such as microscopy, culture and biochemical tests. Antibiotic susceptibility testing was done by Kirby-Bauer disc diffusion technique for the aerobic isolates. The total number of isolates obtained in this study was 210. One hundred and thirty seven (65.5%) were Gram negative, fifty three (25.5%) were Gram positive and twenty (9.5%) were anaerobes. The most frequently isolated organism in this study was Escherichia coli (32.1%), and the least was Enterobacter spp (1.57%) for the aerobes. For the anaerobes, Peptococcus spp (15%), Peptostreptococcus spp (40%), Bacteroides spp (30%) and Fusobacterium spp (15%). The percentage of isolates producing Extended Spectrum Beta-lactamse (ESBL) among E.coli isolates was 44%, with Proteus vulgaris 4% as the least. Percentage of biofilm forming organisms were E.coli (36.8%), S.aureus (23.1%), and Proteus vulgaris 4.2%. E.coli was sensitive to Ciprofloxacin at 57.3% while S. aureus was sensitive to Ofloxacin (63%). Enterococcus faecalis was 100% sensitive to Streptomycin while Enterobacter spp was 100% sensitive to Ofloxacin. No methicillin resistant S. aureus was encountered. The AmpC producers encountered were Klebsiella pneumonia 10% and E.coli 8.1%. The plasmid profile of 15 resistant isolates were studied using the Alkaline lysis method and Gel Electrophoresis. Subsequently, the plasmid analysis and curing using ethidium bromide at various concerntrations were performed. The isolates were exposed to the antibiotics for which they demonstrated resistance initially and the findings revealed nine isolates had plasmids thus suggesting that the mode of antibiotic resistance may be plasmid-mediated. Proper management of diabetic wound infection with appropriate antibiotic therapy should be encouraged.
TABLE OF
CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List of
Tables ix
Figure
x
Abstract xi
CHAPTER 1: INTRODUCTION
1.1 Aims and Objectives 8
CHAPTER 2: LITERATURE REVIEW
2.1 Infections
Associated with Diabetes Mellitus 9
2.1.1 Skin and soft tissue infections 9
2.1.2 Urinary infections 16
2.1.3 Gastrointestinal & liver infections 17
2.1.4 Respiratory infections 20
2.1.5 Head and neck infections 21
2.2 Diabetic Ulcer
23
2.2.1 Etiology
22
2.2.2
Altered Metabolism 25
2.3 Drug
Resistance
28
2.3.1
Types of resistance 27
2.3.5 Multidrug resistant staphylococci 35
2.3.6 Multidrug resistant enterococci 37
2.3.7 Extended spectrum Beta-Lactamases 38
2.4 Biofilm Formation 40
2.5 Mobile Bacterial Genetic Elements 42
CHAPTER 3: MATERIALS AND METHODS
3.1 Study Location 44
3.2 Study Population 44
3.3 Procedure
for the Collection and Processing of Pus 45
3.4 Procedure for Anaerobic Culture
45
3.5 Identification of Organisms 45
3.6 Biochemical Tests 46
3.7 Antimicrobial Susceptibility Testing 48
3.71 ESBL screening and confirmation 49
3.72 Procedure for AmpC 49
3.73 Procedure
for MRSA 50
3.8 Procedure for Biofilm 51
3.8.1 Plasmid
extraction 51
3.8.2 Plasmid analysis 51
3.9 Plasmid curing 52
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Results 54
4.2 Discussion
68
CHAPTER 5: CONCLUSION AND
RECOMMENDATION
5.1 Conclusion 73
5.2 Recommendations
74
References
75
Appendices
91
LIST
OF TABLES
|
4.1
|
Age and
sex distribution of diabetic ulcer patients
|
56
|
|
4.2
|
Distribution
of Bacterial Isolates
|
57
|
|
4.3
|
Distribution
of Anaerobic Organisms
|
58
|
|
4.4
|
Distribution
of Organism With Biofilm Formation
|
59
|
|
4.5
|
Distribution
of ESBL
|
60
|
|
4.6
|
Distribution
of AmpC producers
|
61
|
|
4.7a
|
Antibiogram
of Gram Negative isolates
|
63
|
|
4.7b
|
Antibiogram
of Gram Positive isolates
|
64
|
|
4.8
|
Distribution
of plasmids and resistant isolates.
|
65
|
|
4.9
|
Distribution
of organisms with cured plasmids and antibiotic resistance
|
66
|
LIST OF FIGURE
1
Plasmid Profile of Isolates 67
CHAPTER 1
INTRODUCTION
Diabetes mellitus (DM) is a common metabolic disorder that is
characterised by hyperglycaemia emerging from defects in insulin secretion and
action (ECDCDM, 1997). Diabetes Mellitus includes Type 1 DM which can be
associated with autoimmune damage of the pancreatic beta cells (Shoback et al., 2001)., Type 2 DM, resulting
from insulin resistance and disorder of insulin secretion and gestational
diabetes the third main form that occurs in pregnant women with no history
of DM (Bell and Hockaday, 1996).
Increased blood glucose level associated with Diabetes and the concomitant
metabolic abnormality may be associated with secondary damage in numerous
organs, especially the kidneys, eyes, nerves and blood vessels (ECDCDM,
1997). It presents with symptoms such as
weight loss, visual impairment, thirst and polyuria and it may lead to a
clinical condition known as Ketoacidosis (Butalia et al., 2016). Consequently it could also lead to retinopathy,
nephropathy and neuropathy. In 2015, the World Health Organization estimated
that as many as 366 million people suffered from diabetes and in 2030 the number
would rise to 552million (World Health Organization, 2015). Diabetes is the
sixth leading reason behind death globally (World Health Organization, 2016).
Obesity, alcoholism, smoking and increased sedentary lifestyles
have contributed immensely to the proliferation of diabetic cases globally
(Tuomi et al., 1997). There is also
substantial proof that reinforces the association of diabetes to a group of
genes. The focus for research over the years has been the identification of
genes contributing to the risk of diabetes (Butler and Joffer, 2012). Unlike Type
1 diabetes which is strongly associated with the MHC genes, type 2 has few
genes with profound and major effect. These genes have been detected and as the
cases of diabetes mellitus proliferate, it becomes imperative to monitor blood
glucose levels and ensure proper dieting and regular exercise. Patients with poor blood
sugar regulation are considered immunosuppressed which is a consequence of the
debilitating effects of elevated blood sugars on the immunological system
(Clement and Susan, 2004).
The immune system is
made up of two classes: innate immunity and adaptive immunity. Innate immunity,
the initial line of defense, is triggered when a pathogen initially presents
itself. This portion of immunity is demonstrated at birth and is non-specific
in its mode of action. Futhermore, it serves the entire immune system by
galvanizing specific cells against pathogen invasion to activate the adaptive
immune system. The innate immune system has physical and chemical mechanisms of
response (Bagdade et al., 1974).
Polymorphonuclear
neutrophils (PMN) are a class of white blood cells that have granules in their
cytoplasm with different morphologies (Breedveld et al., 2017). The procedure involved in pathogen suppression are:
(1) PMN cleaveage to vascular
endothelium by the help of the cell surface adhesion molecule L-selectin and
then integrins,
(2) migration and
navigation through vessel wall towards a chemotactic gradient.
(3) phagocytosis and
microbial killing. Although an increase in PMN adhesion to vascular endothelium
has been documented in patients with diabetes, the significance of this change
in mediating a predisposition to infection is unclear (Nolan et al., 1978).
Adaptive immunity is a
specific facet of a properly functioning immune system that gives protection against
previous infections suffered by the host (Holmskov et al., 2003). These responses are mediated by lymphocytes, which
consist of natural killer (NK) cells, B cells and T cells. Vaccinations and
exposure to pathogens benefit the adaptive immune system by establishing
immunologic memory which ensures that the same antigen is contained effectively
on subsequent exposure (Rajiu and Raju 2010). Risk of infection is inevitable
when the pathogen evades the innate immune system machinery and infects the
host. Increased blood glucose causes other disturbing changes in the function
of the immune system such as reduced complement response, leukocyte cleaveage
and bactericidal activity.
Hyperglycemia affects the overall immunity
through different mechanisms. Elevated blood sugar levels in diabetic patients
could lead to acidosis, which compromises the activity of the immune system
(Hill et al., 1974). Chronic hyperglycemia
impedes movement through blood vessels, causing irreparable damage to the nerve
as time progresses (Eriksson et al.,
1989) Hence, the skin, one of the major boundaries in innate immunity, is not
capable of protection against trauma and inflammation due to the presence of
these damaged nerves and this makes the host unaware of any trauma until an
infection emerges. Consequently, skin and soft tissue infections abound in
diabetic patients with increased blood glucose level. Along with poor management
of diabetes, cellulitis and diabetic foot ulcers have difficulty healing and
this could lead to chronic conditions such as osteomyelitis (Schaper and Nabuurs-Franssen, 2002).
Such conditions are properly managed by isolating the
causative organisms and subjecting them to sensitivity test to ensure that the
right antibiotic is dispensed for effective treatment. It is also important to
ensure proper wound care by a competent medical staff. Damaged nerves can be seen all over the body
including the urinary tract. With damage to the nerves in the urinary tract,
urine retention will inevitably encourage the emergence of urinary tract
infections (Stevens et al., 2012).
Although the most common
infections in diabetic patients involve the skin and urinary tract, more severe
infections may arise if blood sugars are not controlled effectively. High
glucose levels limit and deregulate neutrophil synthesis, which is essential in
the immune system to attack a foreign object (Davey et al., 2005). Cytosolic calcium in polymorphonuclear leukocytes
(PMNs) becomes elevated in the presence of hyperglycemia and is inversely
proportional to the occurrence of phagocytosis in patients with type II
diabetes. High levels of cytosolic calcium inhibit the synthesis of adenosine
triphosphate (ATP), which is essential for phagocytosis (Gough et al., 1997). The ability of PMN
leukocytes to mobilize to the site of infection and stimulate apoptosis is
severely compromised.
The ability of bacteria
to survive in the presence of hyperglycemia activates the immune response to
fight such infections. Futhermore, a hyperglycemic state negatively affects the
body’s ability to respond to antimicrobial therapy (Mowar and Baum, 1971).
Common bacterial infections include those caused by Gram negative organisms
such as Pseudomonas aeruginosa,
Klebsiella pneumoniae, and E. coli.
Gram-positive organisms, such as Staphylococci and Streptococci are common, also. Anaerobic organisms may be
present due to decreased blood and oxygen movement into the blood vessels for
the synthesis of leukocytes. Other infections include fungal infections such as
Candida, and viral infections (Muller et
al., 2005).
Holistically, infectious diseases are predominant and chronic in
patients with diabetes mellitus which consequently increases their mortality. A
larger part of these diabetic infections is caused by the hyperglycemic environment
that encourages immune system dysfunction example; neutrophil malfunction,
neuropathy, depression of the humoral immunity and antioxidant system, macro
and micro angiopathies, decrease in antibacterial activity of urine, gastrointestinal and urinary dysmotility (Flemming et al., 2014).
Foot ulcers are a serious clinicopathological outcome of diabetes
with recent studies revealing that life time risk of developing a foot ulcer in
diabetic patients could be as high as 25% (Ahmed
and Choudhury, 2006). They lead to proximate and
non-traumatic causes of leg amputation (Lipsky, 2004). The prevalence of foot
infections in persons with diabetes ranges from a lifetime risk of up to 25% in
all persons with the diagnosis, to 4% yearly in patients treated in a diabetic
foot center (Singh et al., 2005).
Diabetic foot infections are characterized by cellulitis and post-traumatic
infections, but results through ulcerations secondary to progressive peripheral
neuropathy. This leads to the loss of preventive sensation, as well as foot
deformities, gait disorders, anterior displacement of weight-bearing during
walking, and reduced mobility (Pataky et al., 2005). These complications are
commonly accompanied by arterial insufficiency and other chronic immunological
disturbances. Various organisms inhabit the wound and in most patients one or
more species of organisms multiply in the wound which may lead inexorably to
tissue damage. Among the bacterial pathogens, Gram positive organisms such as Staphylococcus aureus and Staphylococcus epidermidis are the most
prevalent in wound infection while Gram negative organisms such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella
species and Proteus species are
rare. Predisposition to urinary tract
infections in Diabetes mellitus results from several factors. High urine
glucose content and defective host immunity are key factors (Patterson and
Androiole, 1997). Due to common or recurrent infections, diabetic patients have
more antibiotic treatments compared with other subjects which can increase the
antibiotic resistance rates in the bacteria (Watters et al., 2014). Early diagnosis is crucial in case of
life-threatening infection (Zhanel et
al., 1991). Due to the presence and increase in antibiotic resistant
bacteria, empirical therapy for severe infections should be later corrected
through in-vitro susceptibility data from the laboratory (Stapleton, 2002).
Therapy by narrow-spectrum antibiotics can prevent the increase in antibiotic
resistance and emergence of multi-drug resistance in diabetic patients
(Rayfield et al., 1982). Presently
there is increasing empirical evidence suggesting that diabetes is a risk
factor for antibiotic–resistant S.pneumonia,
MRSA with basal sensitivity to vancomycin and vancomycin-resistant
enterococci as well as ESBL producing Gram negative bacteria and carbapenem-resistant
Pseudomonas spp followed by Acinetobacter species.
Methicillin-resistant Staphylococcus
aureus (MRSA) is a group of Gram
positive bacteria that are genetically distinct from other strains of Staphylococcus aureus. They come into
existence through horizontal gene transfer, multiple drug resistance to
beta-lactam antibiotics and natural selection (Gurusamy et al., 2013). Extended Spectrum Betalactamses (ESBL) are enzymes
capable of breaking down penicillins, broad-spectrum cephalosporins and
monobactams. They are seen on plasmids that are transferable from strain to
strain and between the same bacterial species (Jacoby, 1997). Comprehensive
evaluation of different aspects of diabetes control aid in the reduction of infection
susceptibility. Literature suggests maintaining blood glucose levels below 200
mg/dL. Glucose levels above 200 mg/dL can lead to increased risk of infections (Kahn
et al., 1996). To aid in the
maintenance of proper perfusion through blood vessels, adherence to standard of
care is very important. Standard of care includes maintaining HbA1c (Glycated
Haemoglobin) < 7%, blood pressure <130/80 mmHg, proper control of lipd
profile levels (Lyudmila and Ivan, 2013).
Reported cases of diabetic infections which investigated the
prevalence of microbes and their associated multi-drug resistance have been
published in developed countries. Contrastingly, the bacteriology of diabetic
ulcers in Nigeria has not been studied extensively especially in some parts of
the country. The alarming fact is Imo State has many diabetic individuals and
the cases are poorly documented and the incidence of foot problems and
amputations cannot be overemphasized. The increased relationship of multi-drug
resistant(MDR) pathogens with diabetic ulcers further compounds the problem
faced by the physician or the surgeon in treating diabetic ulcers without
resorting to amputation (Yoga et al., 2006).
Infection with MDR pathogens incurs a lot of expenses and leads to delayed
hospital stay, and sometimes increases the chances of mortality in diabetic
patients. Careful selection of antibiotics based on the susceptibility pattern
of the isolates from the lesions is most realiable for the proper management of
these infections. Presently, there is limited data on ESBL-producing organisms
from diabetic foot infections especially in this part of the world. The
territorial prevalence of T2DM in Nigeria has been reportedly variable across
different areas of the country which could be a manifestation of cultural,
tribal and food values and lifestyles (Gabriel et al., 2015). In Nigeria, the prevalence of T2DM was calculated to
be 2.2% by the Expert Committee on non-communicable diseases in 1997 with
highest prevalence of 7% in Lagos Island and 0.5% in Mangu, Plateau State
(ECDCDM, 1997). In the 2013 International Diabetic Foundation global study, a
prevalence of 5% was estimated for Nigeria, accounting for 3.9million cases
among persons aged 20–79 years (Guariguata et
al., 2014); 3.6% was reported in Abia State South East non-communicable
diseases survey (Okpechi et al.,
2013). These statistical findings offers a strong basis for investigation into
the pathology of diabetic ulcers hence, the study was designed to determine the
bacteriological profile of diabetic ulcers in Imo State and their in vitro
susceptibility to routinely used antibiotics. The prevalence of multidrug
resistant pathogens in patients with diabetic ulcers was also studied.
1.1 AIM
The aim of this research was to establish the common bacterial
pathogens associated with diabetic ulcers in Imo State and determine the
antibiogram. The generated data could form basis for antibiotic prescriptions
before culture results are obtained. This will be of local clinical relevance
that would provide clinicians with therapeutic options.
Objectives
- To isolate and
identify organisms associated with diabetic ulcers.
- To obtain the
antibiotic sensitivity pattern of various isolates.
- To identify ESBL
(Extended Spectrum Beta lactamase) producers, biofilm forming organisms,
AmpC producers, plasmid profile of isolates, methicillin resistant S. aureus and anaerobic organisms
associated with diabetic ulcers.
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