ANTIBIOTICS SUSCEPTIBILITY PATTERN OF DIFFERENT BACTERIA ASSOCIATED WITH WOUND SEPSIS

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TABLE OF CONTENT

Tile Page                                                                                                         i

Approval Page                                                                                                ii

Dedication                                                                                                      iii

Acknowledgment                                                                                           iv

Table of Content                                                                                             v

List of Figures

List of Table                                                                                       

Abstract

CHAPTER ONE

1.0       Introduction                                                                                        1

1.1       Problem of Statement                                                                         13                              

1.2       Justification                                                                                        14   

1.3       Aim and Objective of the study                                                         15

CHAPTER TWO

2.0       Literature Review                                                                               17       

CHAPTER THREE

3.0       Materials and Method                                                                         29

3.1       Sterilization of Materials                                                                     29

3.2       Sample Size                                                                                         29

3.3       Collection and Processing Sample                                                      30

3.4       Culture Media                                                                                     30

3.5       Isolation of Cultures                                                                           31

3.6       Characterization of Bacteria Isolates                                                  32

3.7       Colonial Morphology                                                                          32

3.8       Cellular Morphology                                                                           32

3.9       Biochemical Characterization                                                             34

3.10     Antibiotics Sensitivity Test                                                                 36

CHAPTER FOUR

4.0       Results                                                                                                39

CHAPTER FIVE

5.0       Discussion                                                                                           60

5.1       Conclusion                                                                                          66

5.2       Recommendation                                                                                67

References

 


LIST OF FIGURES

Figure 1:          Frequency of Occurrence of bacterial from Wound Infection patient in University of Ilorin Teaching Hospital.

Figure 2:          Bacterial Isolate from patient with diabetic ulcers in University of Ilorin Teaching Hospital.

Figure 3:          Bacterial Isolated from patients with surgical wound in University of Ilorin Teaching Hospital.

Figure 4:          Bacterial Isolated from patients with burns in University of Ilorin Teaching Hospital.

Figure 5:          bacterial Isolated from patient with pressure sore in University of Ilorin Teaching Hospital.


LIST OF TABLE

Table 1:           Morphological Characteristics, Staining Reaction and Biochemical Test

Table 2:           Frequency of Occurrence of bacteria from Wound Infection patient in University of Ilorin Teaching Hospital.

Table 3:           Bacteria pathogens causing wound infection in patients with Diabetic ulcers.

Table 4:           Bacteria pathogens causing wound infection in patients with Surgical Wound.

Table 5:           Bacteria pathogens causing wound infection in patients with Burns.

Table 6:           Bacteria pathogens causing wound infection in patients with Pressure Sore.

Table 7:           Antibiotic susceptibility Patterns of Staphylococcus aureus isolated from wound patients in UITH

Table 8:           Antibiotics susceptibility pattern of Pseudomonas aeruginosa isolated from wound in UITH

Table 9:           Antibiotic susceptibility pattern of Staphylococcus epidermidis in UITH.

Table 10:         Antibiotic Susceptibility pattern of Escherichia coli isolated from wound patients in UITH.

Table 11:         Antibiotic Susceptibility pattern of Streptococcus pyogenes isolated from wound patients in UITH.

Table 12:         Antibiotic Susceptibility pattern of Klebsiella pneumoniae isolated from wound patients in UITH.                                         

 

 

                                                 

 

 

 

 

 

 

 


ABSTRACT

This work investigated the antibiotic susceptibility profile of bacterial associated with wound sepsis of patients attending University of Ilorin, Ilorin, Teaching Hospital. Wound swabs were collected from a total number of Hundred patients with different kinds of wound (surgical wound, burn, ulcers and pressure sores) and cultured, of which 72 samples showed bacterial growth. Six different species of bacteria were isolated. Staphylocococcus aureus (47.2%) Pseudomonas aeruginosa (19.5%), Klebsiella pneumonia (6.85%), Escherichia coli (15.3%), Staphylococcus epidemidis (5.6%) and Streptococcus pyogenes (5.6%). The antibiotics susceptibility test of these bacterial isolate was performed using the Kirby- bauer dist diffusion method. Gantamycin and Sreptomycin shows high effectiveness to all the isolate except Staphylococcus epidermis and Klebsiella with pefloxacin and ceftadine respectively showing sensitivity to them. Resistance were shown amongst Ampicillin, contrimoxazole and Tetracycline. This study has revealed Gentamycin as the only antibiotic to which most bacterial isolate from infected wounds were sensitive to. Ampicillin and penicillin were effective against Streptococcus pygenes while zinacef and ceftazidime shows high effectiveness against Klebsiella pneumonia.

KEYWORDS: wound infection: antibiotics susceptibility profile: staphylococcus aureus: gentamycin.


CHAPTER ONE

1.0  Introduction

             A wound results following disruption of the skin which can be intentional or accidental (Giacometti, 2000).Wound infections cause a burden of disease and morbidity for both the patient and the health services. To the patient it causes pain, discomfort, inconvenience, disability, financial drain, and even death due to complications such as septicemia. It causes financial strain on the health services due to the required high cost of hospitalization and management of the patients.

             A number of factors contribute to wound infection; however microorganisms are the major cause with bacteria being the most prevalent (Obuku, 2012). Early recognition of wound infection and appropriate management is important. Antibiotic therapy and surgical management are the cornerstone measures whereby antibiotics offer adjuvant treatment. Wound infection can be caused by single bacteria or multiple microorganisms. Surgical site infections are the second most common cause of nosocomial infections after urinary tract infections (Perencevich,2003)              Most surgical site infections occur in ambulatory patients after discharge from the hospital and

therefore beyond the hospital infection control surveillance programs (Cosgrove, 2003). Prolonged preoperative hospital stay and exposure to diagnostic procedures has been associated with increased rate of surgical site infection. In clean surgical procedures, Staphylococcus aureus is the most common pathogen while Pseudomonas aeruginosa is the most common gram negative bacilli.

            A number of studies indicate an increase in antibiotic resistant microorganisms in surgical patients. Resistant bacteria causes severe infections that are expensive to diagnose and difficult to treat. The mechanism by which resistance develops is complex and can result in multi-drug resistant bacterial strains due to simultaneous development of resistance to several antibiotics. Determination of local bacterial sensitivity patterns to antibiotics is important in providing a guide for antibiotic selection.

            There are factors that increase the risk of wound infection which include patient characteristics such as; age, obesity, malnutrition, endocrine and metabolic disorders, smoking, hypoxia, anaemia, malignancies and immunosuppressants (WHO, 2007). Other factors are the state of the wound which includes nonviable tissue in the wound, foreign bodies, tissue ischaemia, and formation of haematomas, long surgical procedures, and contamination during operation, poor surgical techniques, hypothermia and prolonged pre-operative stay at the hospital.

             Wound infections can be prevented by restoring blood circulation as soon as possible relieving pain, maintaining normal body temperature, avoiding tourniquets, performing surgical toilet and debridement of the wound as soon as possible, administration of antibiotic prophylaxis for deep wound and high risk infections (WHO, 2007). High risk wounds include contaminated wounds, penetrating wounds, abdominal trauma, compound fractures, wounds with devitalized tissue; high risk anatomical sites such as hands and feet. Antibiotic prophylaxis should be started two hours before the surgical procedures.

            Establishment of the causative microorganism is important and treatment should be initiated based on the bacterial sensitivity patterns. Topical silver dressings have been used to treat infected wounds however; there is no evidence for their efficacy due to multiple microbial aetiologies. (Vermenlen, 2007). To achieve optimum antimicrobial therapy, the biofilm load should be reduced to enhance drug concentration at the wound site (Strup et al, 2013).

 

             Bacterial wound infections are a common finding in open injuries. Severe and poorly managed infections can lead to gas gangrene and tetanus which may cause long-term disabilities (Bjarnsholt, 2013). Chronic infection can cause septicemia or bone infection which can lead to death. Sepsis associated encephalopathy increases morbidity and mortality especially in the ICU patients (Maramattom, 2007).

             Infection is an important cause of morbidity and mortality in hospitalized burn patients in patients with burn over more than 40% of the total body surface area, 75% of all deaths following thermal injuries are related to infections (Vindence,2005). The rate of nosocomial infections is higher in burn patients (WHO, 2007)due to  various  factors like nature of burn injury itself, immunocompromised  status of the patient (Preuitt,2008), age of the patient, extent of injury, and depth of burn  in combination  with microbial factors such  as type and  number of organisms, enzyme and  toxin  production, colonization  of the burn wound site, systemic dissemination of the colonizing organisms(Preuitt,2004). Moreover the larger area of tissue is exposed for a longer time that renders patients prone to invasive bacterial sepsis. In extensive burns when the organisms proliferate in the eschar, and when the density exceeds 100,000 organisms per gram of tissues, they spread to the blood and cause a lethal bacterenia. Therapy of burn wound infections is therefore aimed at keeping the organisms’ burden below 100,000 per gram of tissues which increases the chances of successful skin grafting.

            The denatured protein of the burn eschar provides nutrition for the organisms. A vascularity of the burned tissue places  the  organisms  beyond  the  reach  of  host  defense  mechanisms  and  systemically  administered  antibiotics  (Canton,2002).  In addition, cross-infection results between different burn patients due to overcrowding in burn wards.  Also  thermal destruction  of  the  skin  barrier  and  concomitant  depression  of  local  and  systemic  host  cellular  and humeral  immune responses  are  pivotal  factors  contributing  to  infectious  complication  in  patients  with  severe  burn  (Maramattom,2007).  Burn wound infections are largely hospital acquired and the infecting pathogens differ from one hospital to another. The burn wound represents a susceptible site for opportunistic colonization by organisms of endogenous and exogenous origin; thermal injury destroys the skin barrier that normally prevents invasion by microorganisms. This makes the burn wound the most frequent origin of sepsis in these patients (Anguzu, 2005). Burn wound surfaces are sterile immediately following thermal injury, these wounds eventually become colonized with microorganisms, gram-positive bacteria that survive the thermal insult, such as S. aureus located deep within sweat glands and hair follicles, heavily colonize the burn wound surface within first 48h (Oduyebo, 2008).                        Topical  antimicrobials  decrease  microbial  overgrowth  but  seldom  prevent  further  colonization  with  other potentially  invasive bacteria and  fungi. Gastrointestinal  and  upper  respiratory  tract  and  the  hospital environment ( Hansbrough,2007). A susceptible site for opportunistic colonization by organisms of endogenous and exogenous origin; Following colonization,  these organisms start penetrating  the  viable  tissue depending  on  their  invasive capacity, local wound  factors and  the degree of the  patient  s  immunosuppression. If sub-eschar  tissue is  invaded,  disseminated infection  is  likely  to  occur,  and  the  causative  infective  microorganisms  in  any  burn  facility  change  with  time Individual organisms are brought into the burns ward on  the wounds of new patients. These organisms then persist in the resident  flora  of  the  burn  treatment  facility  for  a  variable  period  of  time,  only  to  be  replaced  by  newly  arriving microorganisms. Introduction of new topical agents and systemic antibiotics influence the flora of the wound (NCCLS, 2000). The aim of the present study was to obtain information about the type of isolates, identification and antimicrobial sensitivity of bacterial wound infections in burn patients.

             Most wound infections can be classified into two major categories; skin and soft tissue infections although they often overlap as a consequence of disease progression. Infections of hospital acquired wounds are among  the  leading nosocomial causes of morbidity a4nd increasing medical expense .Routine surveillance for hospital acquired wound infection is recommended by both the centers for disease control and prevention and surgical infection  society .

            The most useful classification of wound from a practical point of view is the rank and wakefield classification (Russell et al., 2004) which classified wounds into tidy and untidy wounds. Tidy wounds are inflected by sharp instrument and contain no devitalized tissue. Examples are surgical incisions, cut from glass, knife and machete. Skin wounds will usually be single and clean cut. Untidy wounds results from crushing, tearing, avulsion, vascular injury or burns and contain devitalized tissue skin wounds will often be multiple and irregular.

            Open wounds (Wikipedia, 2007) can be classified into a number of different types according to the object that caused the wound. Types of open wound include: incision or incised wounds, laceration, abrasions (grazes) puncture wounds and gunshot wounds, penetration wounds and gunshot wounds. Closed wounds have fewer categories but are just as dangerous as open wounds. The types of closed wounds include: contusion, hematoma, crushing injuries. Bruise, contusion and Hematoma: a closed blunt injury may result in a bruise or contusion. There is bleeding into the tissue and visible discoloration where the amount of bleeding is sufficient to create localize collection in the tissue, it is described as hematoma.

Puncture wounds and Bites: puncture wound is an open injury in which foreign materials and organisms are likely to be carried deeply into the underlying tissues. A major danger is that they may give rise to an abscess. They are caused by an object puncturing the skin such as nail or needle.

Abrasion and Friction Burn: is a shearing injury of skin in which the surface is rubbed off. Most are superficial and will heal by epithelization but some may result in full thickness skin loss. It is often caused by sliding fall unto rough surfaces.

Laceration: is the result of which a sharp object causing irregular wounds to soft tissues which lie over hard tissue.

Traction and Avulsion: Avulsion injuries are open where there has been a severe degree of tissue damage such injuries occur when hands or limbs are trapped in moving machinery such as rollers.

War wounds and Gunshot injuries: are associated with different severity of tissue damage depending on whether the injury is of low or high velocity.

Crush: are further variant of blunt injury and are often accompanied by degloving and compartment syndrome (Wikipedia, 2007).

Ulcer: is any breach in an epithelial surface. Chronic ulcers (Example is leg ulcer seen in diabetes mellitus patient) are wounds that fail to heal.

Pressure sore: is also known as decubitus ulcer. These are chronic wounds following tissue necrosis from pressure. They occur over bony prominences. They arise where there is unrelieved pressure in the soft tissue overlying bone such that the external pressure exceeds capillary perfusion and ischemic necrosis occurs.

Burns: are tissues from thermal (heat or cold) application or from the absorption of physical energy or chemical contact. Types include scalds, fat burn, flame burns, electrical burns, cold injury, friction burns, cognizing radiation and chemical burns (Russell et al.,2004)

            The cell wall of bacteria consists of toxic lip polysaccharides called end toxins. Many of these bacteria endotoxin are bactericidial or bacteriostatic to related species of bacteria, but not themselves. The concept of that substances derived from organisms may kill another (Antibiosis) is almost as old as the science of microbiology (Bud, 2005). More than 3000 years ago ancient people stumbled over the discovery that some India of Central America use mould to treat rashes and infected wounds.

            In the 1860s, Louis Pasteur showed that many diseases were caused by bacteria. Later he discovered that he may be able to fight germs with other miocrobes. It was two German doctors who were the first to make an effective medication from microbes; Rudolf Emmerich and Oscar Low conducted their experiment in the 1890s. They proved that the germs that would cause one diseases may be cure for another. A medication called pyocyanase was created from Bacillus pyocyaneus and it was effective in killing the organism that caused cholera, typhoid, diphtheria, and anthrax. It was the first antibiotic to used in hospital.

            The antibiotic properties of penicillin spp. were first described in France by Ernest Duchesne in 1897. However, his work went by without much notice from the scientific community until Alexander Fleming’s discovery of penicillin. Modern research on antibiotic therapy began in Germany with the development of the narrow-spectrum antibiotic salvarasan by Paul Ehrlich in 1909 for the first time allowing an efficient treatment of the widespread problem of syphilis. The drug was also an effective against other spirochaetal infected. The invention of the sulfa drug followed that of penicillin. Sulfa drug comes from prontosil. Which is a substance used as a dye. It has been proven that this drug could cure pneumonia, scarlet fever and blood poisoning, (Berkow, 1999). In the 1930s other laboratories started making other drugs in the same family. An American, Doctor Selman Waksman discovered a drug called streptomycin. It originated from microbes found in soil and was a cure for many intestinal diseases. Auremycin a broad spectrum antibiotic was discovered and it was found that it could do the job of both penicillin and streptomycin. Another laboratory discovered chloromycin. In 1949, another laboratory came up with one of most effective antibiotics ever found, Tetracycline. This drug could be used against many bacteria diseases (Skerrft and Stratton, 2006).

             Antibiotics can be obtained from microorganisms, plants or could he synthetically produced, an antibiotic is an antimicrobial substance produced by a living micro-organism and active in high dilution (Thomas, 1988). It was defined as a metabolism product of one organism that is detrimental or inhibitory to other organism in very small amount by Daniel in 1992. Antibiotic is a product of the metabolism of a micro organism that is inhibitory to other micro organisms. Antibiotics was originated referred only to substance extracted from fungus or other organisms but it has later come to include many synthetic and semi-synthetic drugs that have antimicrobial or antibacterial effects (Wikipedia, 2007)

            The effectiveness of antibacterial agents is influenced by a number of factors. The concentration if it will be bacteriostatic or bactericidal. The d\time of exposure and type of organism; the longer the contact time, the greater would be the number of organisms killed.            Organisms which clump together take a longer time to kill than organisms that occur once. Temperature can also influence antimicrobial activity.

            Antibiotics may be categorized (Brooks et al., 2003) according to their   spectrum of activity or  mode of action; Broad (affect both Gram Positive and Gram Negative),Narrow(effect against only one group of organism) or Limited(effective against only one or very few bacteria).They may be classified on the basis of their mode of action(ability to interfere with protein synthesis, structure and function of cytoplasmic membrane, DNA synthesis, or inactive of metabolic pathways). On the basis of their chemical structures they are classified as macrolides (erythromycin), carbohydrate containing antibiotic (aminoglycosides), aromatic antibiotic (chloraphenicol), quinines (tetracycliune), etc.

             Resistance to antibiotic is achieved by the pathogens through the following way (CDC, 2004;Prescott et al 2003)                                                                                                                                            

a) By pumping the antibiotic out of the cell after it has entered.this pumps is known as multidrug –resistance pumps.pathogens with this pumps are Escherichia coli, Pseudomonas aeruginosa and Staphylococcaus aureus.                                                                                                                                       

b) Some pathogen act by inactivating antibiotics through chemical modes. Examples include penicillin’s enzyme action on beta lactam ring of penicillins, phosphorylation of amino glycosides and acetylating of amino glycoside and chloramphenicol.

c) Another way in which resistance to antibiotic is achieved is through the modification of the target sites in the pathogen mutation. This is exhibited in streptomycin resistance by Escherichia coli.

d) Some bacteria lack target structures which antibiotics can attack. An example is seen in Mycoplasma which lack bacterial cell wall hence their resistance to penicillin’s.

e) By preventing entrance of the antibiotic into the cells. This is seen in penicillin resistant gram negative pathogens.

             The gene responsible for antibiotic resistance is located on the plasmid and chromosome in the pathogen (CDC, 2007).

 1.1 Problem Statement

            Septic wounds are a common cause of morbidity. Despite improvement in the practice of medicine and attempts to provide aseptic conditions in the surgical wards, the incidences of wound infection are increasing. Management of wound infection remains a challenge in the surgical areas with the increasing resistance to antimicrobials Antimicrobial resistance can lead to complications which depend on severity that cause disability or death and increased cost of hospitalization and management. In children, this impacts negatively on the quality of life at a tender age. The antibiotic sensitivity patterns have not been studied fully especially in the surgical pediatric patients at University of Ilorin Teaching Hospital. Inappropriate antimicrobial use is associated with increased resistance. It was therefore important to identify the causative organisms and determine the antimicrobial sensitivity patterns to help reduce infections and ensure appropriate use of antimicrobials.

Purpose of the Study

            Wound infection is a common problem in children, proper management with appropriate antibiotics is therefore important to reduce morbidity that may arise. This study aimed to determine the aetiology and antimicrobial sensitivity patterns of bacteria that cause wound infections in the surgical paediatric patients at University of Ilorin Teaching Hospital. Antibiotic misuse and overuse can lead to resistance which necessitated the need for the study.

1.2       Justification

            Wound is considered one of the major health problems in the world and infection is one of the most frequent and severe complications in patients who have sustained wounds.  

            This study is based on understanding that bacteria are responsible for delayed wound healing and the so called normal flora of the skin can become pathogenic. This study will not only identify the bacteria but also perform antibiotics susceptibility test on each them so as to know the drug that will be effective against a particular bacteria present in the wound.

1.4 Aims and Objective

            The main objective of this study was to identify the bacteria that cause wound sepsis and their sensitivity patterns in eastern and western part of Ilorin at the University of Ilorin Teaching Hospital.

Specific Objectives

            The specific objectives were:

  • To determine the prevalence of wound infection in the paediatric surgical wards in University of Ilorin Teaching Hospital.
  • To identify the bacteria that cause wound infection.
  • To determine the antimicrobial sensitivity patterns of the isolated bacteria
  • To find out the antibiotics used in management of wounds in the paediatric surgical wards in University of Ilorin Teaching Hospital.

Significance

            The findings of the study was to help in choosing appropriate antibiotics by considering the sensitivity patterns that were observed, hence appropriate management of the infected wounds.  

            This would result in a cost effective therapy for the patient and reduced financial burden of hospitalization.

 

Limitations

            Wound infections are caused by a number of microorganisms which include bacteria, fungi and viruses. This study was only able to address sepsis caused by aerobic bacteria and anaerobic bacteria. The study was not able to address the factors that contribute to resistance patterns that were observed.     

 

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