ISOLATION AND IDENTIFICATION OF PATHOGENIC BACTERIA FROM DIABETIC ULCER IN ABA METROPOLIS

ABSTRACT

Diabetic foot ulcer is a major complication of diabetes mellitus. The present study was undertaken to evaluate the microbial profile of diabetic foot ulcer in patients in Aba metropolis. Swap samples were collected from twenty patients with diabetic foot ulcers and all yielded growth of bacteria organism. Samples were processed by standard microbiological methods such as microscopy, culture and biochemical test. Antibiotic susceptibility testing was done by disc diffusion technique. The total number of isolates obtained in this study was 42.While 30(71.3%) were gram negative and 12(28.5%) were gram positive. The most frequently isolated organism in this study was Escherichia coli (40.4%) followed by S. aureus and Proteus (19.0%) each, Pseudomonas aeruginosa (7.1%), Strept. spp, klebsiella and COANS (4.7%). The percentage of extended spectrum beta lactamases (ESBL) among E.coli isolates was 35.2% and its percentage among all the gram negative organism was 20%. Almost all the isolates showed resistance against most of the narrow spectrum antibiotics tested like Cotrimoxazole, Chloramphenicol and Amoxillin. No methicillin resistance S. aureus was encountered. Proper management of diabetic wound infection with appropriate antibiotic is very important.

TABLE OF CONTENT

Cover page

Title page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                              iii

Acknowledgment                                                                                                                   iv

Table of content                                                                                                                      v-vi

List of tables                                                                                                                           vii

Abstract                                                                                                                                  viii

CHAPTER ONE

1.0 Introduction                                                                                                                      1

1.1 Aim of the study                                                                                                               3

1.2 Objectives of the study                                                                                                     3

CHAPTER TWO

2.0 Literature review                                                                                                              4

2.1 Risk factors associated with diabetic ulcer                                                                      5

2.2 Pathophysiology                                                                                                               5

2.3 Altered metabolism                                                                                                          7

2.4 Impaired no synthesis                                                                                                       8

2.5 Structural and functional changes in fibroblasts                                                              8

2.6 Increased matrix metalloproteinase (MMP) activity                                                       9

2.7 Footwear                                                                                                                           10

CHAPTER THTREE

3.0 Materials and methods                                                                                                      11

3.1 Collection of samples                                                                                                       11

3.2 Sterilization of materials                                                                                                  11

3.3 Preparation of media                                                                                                        11

3.4 Cultivation of isolates                                                                                                       11

3.5 Subculturing of the isolates                                                                                              12

3.6.0 Identification and characterization of isolates                                                               12

3.6.1 Gram staining                                                                                                                12

3.6.2 Motility test                                                                                                                   13

3.6.3 Biochemical test                                                                                                            13

3.6.3.1 Catalase test                                                                                                                13

3.6.3.2 Urease test                                                                                                                  13

3.6.3.3 Indole test                                                                                                                   14

3.6.3.5 Methy red-Voges Proskauer test                                                                                14

3.6.3.6 Citrate utilization test                                                                                                 14

3.6.3.7 Tripple sugar iron agar                                                                                               15

3.6.4 Sensitivity                                                                                                                      15

3.6.5 Procedure for extended beta lactamases                                                                        16

3.6.6 Phenotypic confirmation test                                                                                         16

CHAPTER FOUR

4.0 Result                                                                                                                                17

4.1 Table 1                                                                                                                              1 7                                                                                                                                    4.2 Table 2                                                                                                                              18

4.3 Table 3                                                                                                                              19

4.4 Table 4                                                                                                                              21

4.5 Table 5                                                                                                                              22

CHAPTER FIVE

5.0 Discussion                                                                                                                         23

5.1 Conclusion                                                                                                                        24

5.2 Recommendation                                                                                                             24

References

LIST OF TABLES

1.Table 1 shows the age distribution of diabetic ulcer patient observed.           17

2. Table 2 shows the frequency and types of isolate observed.             18

3. Table 3 shows samples with single and multiple infections among diabetic wound ulcers.     19

4. Table 4 shows the antibiotics of susceptibility pattern of diabetic wound isolates from Patients.                    20 

5.Table 5 shows ESBL producers among gram positive bacteria isolates.                                   21

CHAPTER ONE

1.0.INTRODUCTION

Diabetes mellitus is a common, chronic debilitating and fatal endocrine disease with constantly growing global prevalence. DM encompasses Type 1 DM which can be associated with autoimmune damage of the pancreatic beta cells. Type 2 DM, resulting from insulin resistance and disorder of insulin secretion and gestation diabetes which can progress to Type 2 DM in the years afterward. In 2001, the World Health Organization estimated that as many as 366 million people suffered from diabetes and in 2030 the number would rise to 552million. (WHO,2015). Generally, infectious diseases are more frequent and serious in patients with diabetes mellitus which potentially increases their morbid-mortality. The greater frequent of infections in diabetes is caused by the hyperglycemic environment that favors immune dysfunction example; damage to the neutrophil function, depression of the antioxidant system and humeral immunity. (Watters et al., 2014)

There is a general consensus among clinicians that diabetic patients are at increased risk of developing infection (Braces, 2007). This special vulnerability has been attributed to impaired leukocyte function associated vascular diseases, poor glucose control and altered host response (McMahon and Bistrian, 1995; Bhatia et al., 2003).

Diabetic foot infections (DFIs) are defined as a clinical syndrome characterized by local findings of inflammation or purulence (sometimes accompanied by systemic manifestations of sepsis) occurring in a site below the malleoli in a person with diabetes. Estimates of the incidence DFIs range from a lifetime risk of 4% in all persons with diabetes to 7% yearly in patients treated in a diabetic foot center (Pecoraro et al., 1990). Most DFIs occur in a neuropathic or neuroischaemic ulcer, which serves as a point of entry for pathogens. With the exception of erysipelas and posttraumatic (including postsurgical) infection (Kay et al., 2012). DFIs are almost always epiphenomena, i.e. the consequence of progressive peripheral polyneuropathy, with associated loss of protective sensation coupled with gait disorders, anterior displacement of weight-bearing during walking (Pataky et al., 2005) with reduced mobility, and arterial insufficiency in a mostly elderly patient population (Pataky and Vischer 2007). Vascular disease, mostly in the form of occlusive atherosclerotic disease of the arteries below the knee, sometimes accompanied by small vessel dysfunction, can cause ischaemic ulcers and may contribute to elevated plantar pressures and to prolonged duration of foot-to-floor contact (Pataky et al., 2003).

Once infection occurs, it is difficult to treat since the clinical course of the infection is more fulminant and severe, and possess a greater threat to the glycemic status of the patient (Louie et al., 1993; Beckert et al., 2006). With the advent of the new strategies and approaches in the prevention of these infections as with the introduction of new insulin preparation for good glycemic control, presumption in the altered patient behaviour may reduce the incidence of infections or alter the type of infection (Eaglstein et al., 1997; Piaggesi et al., 2007). There are several well accepted predisposing factors that place patients with diabetes at high risk for a lower extremity amputation. The most common components in the causal pathway to limb loose include peripheral neuropathy, ulceration, infection and peripheral vascular disease (Armstrong et al., 1998). The development of wounds is a serious complication for patients with diabetes. Numerous factors related to diabetes can impair wound healing, including wound hypoxia (inadequate oxygen delivered to the wound) infection, nutrition deficiencies, and the disease itself (Lavery et al., 2007). Fluctuating blood sugar and hypoxia from poor circulation may impair the ability of white blood cells to destroy pathogenic bacteria and fungi, increasing infection risk (Stadelmann et al., 1998).

A diabetic foot ulcer is an open sore or wound that can be seen in 15% of diabetic patients and it is normally located on the bottom of the foot. Most of the patients with foot ulcers are hospitalized (APMA, 2016). Foot ulcers are a serious complication of diabetes with recent studies suggesting that life time risk of developing a foot ulcer in diabetic patients may be as high as 25%. (Aragao et al.,2010). They are now the most common proximate and non-traumatic causes of leg amputation. (Lipsky et al., 2004).  Various organism colonized the wound and in some patients one or more species of organisms proliferate in the wound which may lead to tissue damage. Among the bacteria pathogens, gram positive organisms such as Staphlococus aureus, Streptococus species and COANS (Coagulase negative Staphs aureus) are the most common in the wound infection while gram negative organism such as Pseudomonas aeruginosa, Escherichia coli, Proteus speciesare also seen.

1.1 AIM OF THE STUDY

The aim of this research study is to evaluate the bacteria diversity of diabetic wound ulcers and documents a baseline rate for antibiotic sensitivity pattern for these organisms which will be of local clinical relevance.

1.2 OBJECTIVES OF THE STUDY

i. To isolate, identify and characterize pathogenic bacteria from diabetic ulcer in Aba metropolis.

ii. To determine the antibiotic sensitivity pattern of isolates.

iii. to determine the prevalence of each isolate.

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