ANTIBIOTIC SUSCEPTIBILITY PATTERNS OF BACTERIA

ISOLATED FROM WARDS, OPERATING ROOM AND POST-

OPERATIVE WOUND INFECTIONS AMONG PATIENTS

ATTENDING MAMA LUCY HOSPITAL, KENYA.

AMULIOTO JOHNSTONE AUNA (BSc, MLS)

P150/28787/2014

A research thesis submitted in partial fulfillment of the requirements for the

award of the degree of Master of Science in Infectious Diseases (Bacteriology) in

the School of Medicine of Kenyatta University.

MAY, 2021

ii

DECLARATION

This thesis is my original work and has not been presented for a degree in any other

university.

Signature: …………………………… Date: ……………………………

Amulioto Johnstone Auna

P150/28787/2014

Department of Medical Laboratory Science

This thesis has been submitted for review with our approval as University Supervisors.

Signature: ………………………….. Date: ………………………….

Dr. Margaret Muturi (PhD),

Senior Lecturer, Department of Medical Laboratory Science

Kenyatta University

Signature: ………………………… Date: ……………………………

Dr. Scholastica Mathenge (PhD),

Senior Lecturer, Department of Medical Laboratory Science

Kenyatta University

iii

DEDICATION

I dedicate this work to my lovely wife and son who have been a pillar in my life.

iv

ACKNOWLEDGEMENT

First of all I thank the almighty God. I do recognize the work of my supervisors Dr.

Margaret Muturi and Dr. Scholastica Mathenge who guided me through the entire

project. Their time and input was valuable in writing the whole document. My sincere

gratitude go to Kenyatta university laboratory technologists Mr. Peter Mugo and Ms.

Ruth Maundu for their technical support. I am grateful to the department of medical

laboratory science for granting me the permission to use the Laboratory for my project.

My heartfelt thanks goes to the administration of Mama Lucy hospital for granting me

access to the facility during data collection. My sincere gratitude’s also go to the staff

of the hospital for creating a favorable research environment. I am really indebted to

my parents and wife who prayed and encouraged me.

v

TABLE OF CONTENTS

TITLE PAGE .................................................................................................................. i

DECLARATION ........................................................................................................... ii

DEDICATION .............................................................................................................. iii

ACKNOWLEDGEMENT ............................................................................................ iv

LIST OF TABLES ........................................................................................................ ix

LIST OF FIGURES ....................................................................................................... x

ABBREVIATIONS AND ACRONYMS ..................................................................... xi

DEFINITION OF TERMS .......................................................................................... xii

ABSTRACT ................................................................................................................ xiv

CHAPTER ONE .......................................................................................................... 1

INTRODUCTION ....................................................................................................... 1

1.1 Background of the study ..................................................................................... 1

1.2 Statement of the problem .................................................................................... 3

1.3 Justification of the study ..................................................................................... 4

1.4 Research questions .............................................................................................. 4

1.5 Objectives ........................................................................................................... 4

1.5.1 General objective ......................................................................................... 4

1.5.2 Specific objectives ....................................................................................... 5

1.6 Significance of the study ..................................................................................... 5

CHAPTER TWO ......................................................................................................... 6

LITERATURE REVIEW ........................................................................................... 6

2.1 Surgical site infection global perspective ........................................................... 6

2.2 The burden of surgical site infections ................................................................. 8

2.3 The problem of antibiotic resistance in surgical site infections .......................... 8

2.4 Risk factors associated with surgical site infections ........................................... 9

vi

2.4.1 Intrinsic factors ........................................................................................ 9

2.4.2 Extrinsic factors ..................................................................................... 12

2.5 Pathogenesis of surgical site infections ............................................................ 14

2.6 Sources of surgical site infections .................................................................... 15

2.6.1 Endogenous sources .................................................................................. 15

2.6.2 Exogenous sources .................................................................................... 16

2.7 Pathogen’s causing surgical site infections ...................................................... 16

2.8 Diagnosis of surgical site infections ................................................................. 19

2.9 Complications of surgical site infections .......................................................... 19

2.10 Treatment and Management of surgical site infections .................................... 19

CHAPTER THREE ................................................................................................... 21

MATERIALS AND METHODS .............................................................................. 21

3.1 Study area ......................................................................................................... 21

3.2 Study design ...................................................................................................... 21

3.3 Study population ............................................................................................... 21

3.4 Patient recruitment ............................................................................................ 21

3.4.1 Inclusion criteria ........................................................................................ 22

3.4.2 Exclusion criteria ....................................................................................... 22

3.5 Sample size determination ................................................................................ 22

3.6 Sampling technique ........................................................................................... 23

3.7 Sample collection and analysis ......................................................................... 24

3.7.1 Patients pus swabs ..................................................................................... 24

3.7.2 Wards and operating room surface swabs ................................................. 24

3.7.3 Microscopy ................................................................................................ 25

3.7.4 Culture ....................................................................................................... 25

3.7.5 Biochemical tests ....................................................................................... 26

3.7.5.1 Catalase test .............................................................................................. 26

3.7.5.2 Coagulase test ........................................................................................... 27

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3.7.5.3 Urease test................................................................................................. 27

3.7.5.4 Oxidase test............................................................................................... 28

3.7.5.5 Indole test ................................................................................................. 28

3.7.5.6 Methyl red and Voges proskauer tests ...................................................... 28

3.7.5.7 Citrate test ................................................................................................. 29

3.7.6 Antibiotic susceptibility testing ................................................................. 29

3.8 Data collection tool ........................................................................................... 30

3.9 Ethical consideration ......................................................................................... 30

3.10 Data analysis ..................................................................................................... 31

CHAPTER FOUR ...................................................................................................... 32

RESULTS ................................................................................................................... 32

4.1 Patients demographics ...................................................................................... 32

4.2 The prevalence of bacteria isolated from surgical site infections ..................... 32

4.3 The antibiotic sensitivity pattern of surgical site infection bacteria ................. 33

4.4 The resistance pattern of surgical site infection bacteria .................................. 36

4.5 The prevalence of bacteria contaminating the wards and operating room ....... 37

4.6 The antibiotic sensitivity pattern of bacteria from the wards and operating

room….. ....................................................................................................................... 38

CHAPTER FIVE ....................................................................................................... 40

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ........................ 40

5.1 Discussion ......................................................................................................... 40

5.1.1 The prevalence of bacteria isolated from surgical sites infections ............ 40

5.1.2 The prevalence of bacteria contaminating the wards and operating room 41

5.1.3 The antibiotic sensitivity pattern of bacteria isolated from surgical site

infection. ................................................................................................................... 42

5.1.4 The antibiotic sensitivity pattern of bacteria from the wards and operating

room…… ................................................................................................................. 44

5.2 Conclusions ....................................................................................................... 45

5.3 Recommendations ............................................................................................. 45

viii

5.4 Recommendations for further research ............................................................. 45

REFERENCES ........................................................................................................... 46

APPENDICES ............................................................................................................ 64

Appendix I-Consent form ............................................................................................ 64

Appendix II-Questionnaire .......................................................................................... 66

Appendix III- KUERC approval letter ......................................................................... 67

Appendix IV- Gram staining technique ....................................................................... 69

Appendix V- Biochemical tests ................................................................................... 70

Appendix VI- Publication abstract............................................................................... 72

Appendix VII- Culture plates ....................................................................................... 73

Appendix VIII- An algorithm for identifying Gram-positive bacteria ........................ 74

Appendix IX - Preparation of MacConkey agar .......................................................... 75

Appendix X- Preparation of Blood agar ...................................................................... 76

Appendix XI- Preparation of Mueller Hinton .............................................................. 77

Appendix XII - Mama Lucy hospital research approval letter .................................... 78

ix

LIST OF TABLES

Table 4.1: The antibiotic sensitivity pattern of Gram-positive bacteria from patients

with surgical sites infections at Mama Lucy Hospital……………………34

Table 4.2: The antibiotic sensitivity pattern of Gram-negative bacteria from patients

with surgical sites infections at Mama Lucy Hospital…………………...35

Table 4.3: The resistance rate of Gram-positive bacteria isolated from surgical site

infections against the drugs tested………………………………………..36

Table 4.4: The resistance rate of Gram-negative bacteria isolated from surgical site

infections against the drugs tested………………………………………..37

Table 4.5: The prevalence of bacteria contaminating the different sections of Mama

Lucy Hospital……………………………………………………………..38

Table 4.6: The antibiotic sensitivity pattern of bacteria recovered from the wards and

operating room of Mama Lucy Hospital………………………………….39

x

LIST OF FIGURES

Figure 3.1: Streaking method………......................................................................... ..26

Figure 4.1: Patients age distribution………………………………………………….32

Figure 4.2: The prevalence of bacteria isolated from patients with surgical site

infection at Mama Lucy Hospital………………………………………..33

xi

ABBREVIATIONS AND ACRONYMS

AIC Africa Inland Church

ASA American society of anesthesiologist

ATCC American type culture collection

BMI Body Mass Index

BSAC British Society for Antimicrobial Chemotherapy

CDC Centre for disease prevention and control

CLSI Clinical and Laboratory Standards Institute

CoNS Coagulase negative staphylococcus

ESBL Extended spectrum beta-lactamase

EUCAST European Committee on Antimicrobial Susceptibility Testing

HPS Health protection Scotland

IMVIC Indole, methyl red, voges proskauer in citrate test

IPC Infection prevention and control

KUERC Kenyatta University Ethics Review Committee

MRI Magnetic resonance imaging

MRSA Methicillin resistant Staphylococcus aureus

MTRH Moi Teaching and Referral Hospital

NHS National Health Service

NHSN National Healthcare Safety Network

POD Post-operative day

SPSS Statistical package for the social sciences

SSI Surgical site infection

USA United States of America

VAC Vacuum-assisted closure

xii

DEFINITION OF TERMS

Antibiotic prophylaxis - Involves the administration of antibiotics to prevent the

development of infection for example SSI.

ASA score - This is a classification system used to measure a patient’s pre-operative

physical condition. Together with other parameters it is used to evaluate the patient’s

risk of acquiring infection.

Debridement - This is the wide removal of unhealthy tissues from a surgical wound to

promote healing of the remaining healthy tissues.

Emplaced - This is when something is put in place for example an implant.

Extrinsic - Features that are external to the patient.

Healthcare associated infection - This is an infection that a patient can get while

receiving treatment in a hospital and the infection was not present during the admission

period.

Intermediate sensitivity - This is a response to an antibiotic elicited by a pathogen that

requires a larger dose than normal to treat the infection.

Intrinsic - These are features present within the patient.

Monomicrobial - It means that one micro-organism (bacteria) was isolated.

Polymicrobial - This means that more than one micro-organism (bacteria) was isolated.

Resistant - This is a non-response to an antibiotic by a pathogen irrespective of the

dose.

xiii

Risk factor - This is a variable associated with the increased chances of getting an

infection for example patients with a higher body mass index (BMI) have a greater

likelihood of getting SSI.

Sensitive - This is a response to an antibiotic elicited by a pathogen when the drug is

administered against it.

Surgical site infection – This is an infection of the post-operative period occurring in

any part of the body following an operation.

Surgical wound - Refers to a wound created by a cutting instrument like a scalpel.

Wound classification - This is a classification scheme for categorizing wounds based

on the degree of bacterial wound contamination at the time of surgery. Together with

other parameters, the system is used to predict the likelihood of the patient developing

surgical site infection.

xiv

ABSTRACT

Surgical site infections are a worldwide problem in the field of surgery contributing to

increased mortality and morbidity. However, despite advances in the control of surgical

site infections, the risk of acquiring these infections has not fully been eliminated due

to the emergence and spread of resistant bacteria pathogens. In Kenya, there were

scanty published reports on the antibiogram of surgical site infection pathogens lurking

in the hospitals. The objective of this research was to determine the prevalence and

antibiotic susceptibility patterns of bacteria isolated from the wards, operating room,

and surgical site infections among patients attending Mama Lucy Hospital. This was a

cross-sectional descriptive study of patients with post-operative wound infections in the

hospital wards. Purposive sampling was employed and a total of 126 samples collected.

Of these, 58 came from surgical site infected patients and 68 were obtained from

predefined areas of the wards and operating room of the facility. The samples were

processed through Gram stain, culture, and an array of biochemical tests. Subsequently,

antibiotic susceptibility tests by Kirby bauer disc diffusion technique were performed

on the isolated bacteria. Data collected was analyzed using a statistical package for the

social sciences (SPSS) version 20 and chi-square (p<0.05). A total of 137 bacteria were

isolated from the culture positive swabs, 78 of these came from the wound pus swabs

while 59 were recovered from the hospital surface swabs. Among the SSI bacteria,

Staphylococcus aureus (28.2%) was the preponderant bacteria followed by E.coli

(15.4%). In the wards and operating room, the main bacterial contaminant was

Staphylococcus aureus. Based on the sensitivity report, all the SSI bacteria isolates

were sensitive to Chloramphenicol (69.2%). Wherease the environmental bacteria

isolates had sensitivity to Ciprofloxacin (86.4%), Doxycycline (88.1%),

Chloramphenicol (93.2%) and Vancomycin (100%). Majority of the environmental

isolates were highly resistant to Ampicillin/cloxacillin. In conclusion, the most

prevalent SSI bacteria was Staphylococcus aureus, while Chloramphenicol was seen as

the best drug for treating SSI at the hospital. The facility therefore needs to identify the

most frequent bacteria associated with SSI. In addition, they need to monitor the

bacteria that frequently contaminate the wards and operating room. The current

antibiogram profile will help policy makers in the healthcare sector and the current

setting to improve the current local guidelines on antibiotic prophylaxis for treatment

of surgical site infection. The profiling will also assist in monitoring bacteria resistance

trends within the institution and the country. Information generated from the hospital

environment will help the infection control team at the current set-up to improve on the

prevention of healthcare associated infections by carrying out active monitoring of

hospital contaminants.

1

CHAPTER ONE

INTRODUCTION

1.1 Background of the study

A surgical site infection (SSI) is an infection that occurs within 30 days after an

operation or within one year if an implant was emplaced. According to the National

Healthcare Safety Network of Center for disease prevention and control (CDC),

surgical site infections are classified based on depth and tissue spaces involved. These

are superficial incisional, deep incisional, or organ/spaces surgical site infections

(Horan et al., 2008).

This infection is known to affect people of all age groups, this is supported by a study

that observed patients with as low as 15-79 years with surgical site infected wounds.

The participants had a mean age of 35.95 ± 19.01 years (Mengesha et al., 2014). The

high SSI incidence rates have been witnessed in males than females and vice versa

(Rahman et al., 2019; Seni et al., 2013).

Surgical site infections accounts for more than one-third of post-operative deaths

worldwide (Fan et al., 2014). In addition, the disease contributes to an increase in

morbidity, prolonged lengths of hospital stay, and increased healthcare costs among

hospitalized patients (Jenks et al, 2014; Roy et al., 2014). In the United States of

America (USA) alone, the total annual expenditure for five major infections was $ 9.8

billion with SSIs and ventilator-associated pneumonia accounting for 33.7% and 31.6%

of the total cost respectively (Zimlichman et al., 2013). Whereas the average cost per

patient for USA hospital inpatient services was $34670 (Scott II, 2009).

In low and middle income countries SSI remains one of the leading healthcare-acquired

infection with incidence rates ranging from 0.4 to 30.9 per 100 patients undergoing

2

surgery and a pooled incidence rate of 11.8% per 100 patients undergoing surgery,

strikingly higher than proportions recorded in developed countries (World Health

Organization, 2011). The situation in these countries has been augmented by the

absence of guidelines on antibiotic stewardship, inadequate guidelines on infection

prevention and control, and the emergence of drug resistant bacteria like Methicillin

resistant Staphylococcus aureus and other resistant bacterial strains (Asaad and Badr,

2016; Allegranzi et al., 2011; Weinshel et al., 2015). Moreover, a research in Ethiopia

on SSI observed that almost 100% of Gram-positive bacteria and 95.5% of Gram-

negative bacteria were resistant to more than two antibiotics (Mulu et al., 2012).

The prevalence rate of SSI antibiotic resistant bacterial strains in the country is

unknown. Besides, there is no data on the prevalence of SSI pathogens and their

antibiograms at the current hospital. This is because the facility lacks a laboratory

bacteriology section for bacterial culture and relies mainly on empiric therapy. To

effectively treat SSI patients, surgeons require information on SSI causative agents and

their antibiograms. However this information is largely missing at the present setting,

as a result, the present study was carried out in order to bridge the existing gap.

Besides intrinsic and procedure related risk factors, a contaminated hospital

environment has been seen to increase the risk of surgical patients acquiring SSI (Fegio

and Afriyie-Asante, 2014). This is because a contaminated environment may serve as

a haven for bacteria proliferation. The consequences of this is surgical site infection in

immune suppressed individuals, which can arise from bacterial wound contamination

during surgery (Atata et al., 2010). The problem can be complicated further by the

emergence of antibiotic-resistant environmental bacterial strains making the treatment

of SSI more costly and difficult (Solomon et al., 2017).

3

To prevent this, active monitoring of environmental pathogens with positive feedbacks

for the infection control team is encouraged (Nedelcheva et al., 2018). Most of these

environmental pathogens vary from one setting to the other while exhibiting unique

antibiotic susceptibility. Hence this project was done to determine the prevalence and

antibiotic susceptibility patterns of bacteria isolated from the wards, operating room

and among SSI patients attending Mama Lucy Hospital.

1.2 Statement of the problem

Despite the progress made in the field of surgery, surgical site infections remain a global

problem accounting for increased deaths and morbidity among hospitalized patients.

Poor infection prevention and control programs (IPC), inadequate guidelines on

antibiotic stewardship, and the emergence of antibiotic-resistant bacterial strains have

augmented the situation making the choice of empirical therapy more difficult and

expensive. In Kenya, the infection rates of surgical site infections from Agakhan

University Hospital and Kenyatta National Hospital stand at 7%, 30.8%, and 37.7%

respectively (Dinda et al., 2013; Miima et al., 2016; Opanga et al., 2017). However,

antibiogram data on SSI causative pathogens which is vital in the management of

surgical site infections is insufficient. This is because some facilities especially public

hospitals lack the laboratory capacity to perform bacterial cultures due to limited

resources.

Surgical patients in these facilities also face the risk of acquiring infections from

contaminated wards and operating room. Most of these environments have their unique

bacterial flora to which patients are at risk of acquiring infections during

hospitalization. These bacterial flora exhibit unique patterns of antibiotic activity during

a certain period. Only when antibiogram data is available, are the clinicians able to

manage surgical site infections effectively.

4

1.3 Justification of the study

In spite of surgical site infection being a problem in Kenya, there is insufficient

information on the implicated pathogens and their antibiotic susceptibility profiles. The

few published reports have not provided a clear picture of the magnitude and extent of

SSI pathogens in a whole hospital setting. Given that understanding, the prevailing

antibiogram and causative agent were vital in antibiotic selection for SSI treatment.

This study generated a current antibiogram report that will aid policy makers in the

health sector in updating the current local guidelines on antibiotics prophylaxis for

treatment of surgical site infection. This report will also assist in monitoring bacteria

resistance trends in the country.

1.4 Research questions

1. What is the prevalence of bacteria isolated from patients with surgical site

infections at Mama Lucy Hospital?

2. What is the prevalence of bacteria contaminating the wards and operating room

of Mama Lucy Hospital?

3. What is the antibiotic sensitivity patterns of the bacteria isolated from patients

with SSIs and those contaminating the wards and operating room of Mama Lucy

Hospital?

1.5 Objectives

1.5.1 General objective

To determine the antibiotic susceptibility patterns of bacteria isolated from wards,

operating room, and post-operative wound infections among patients attending Mama

Lucy Hospital, Kenya.

5

1.5.2 Specific objectives

1. To determine the prevalence of bacteria isolated from patients with surgical site

infections at Mama Lucy Hospital.

2. To determine the prevalence of bacteria contaminating the wards and operating

room of Mama Lucy Hospital.

3. To determine the antibiotic sensitivity patterns of the bacteria isolated from

patients with surgical site infections and those contaminating the wards and

operating room of Mama Lucy Hospital.

1.6 Significance of the study

The present study provided the current prevalence of SSI bacteria and environmental

bacteria isolates along with their antibiograms. This information will aid policy makers

in the healthcare sector and the current setting to improve the current local guidelines

on antibiotic prophylaxis for treatment of surgical site infection. The antibiograms will

also assist in monitoring bacteria resistance trends within the institution. The

information generated from the hospital environment will inform the infection control

team at the current setting on areas that require improvement in terms of prevention of

surgical site infections.

6

CHAPTER TWO

LITERATURE REVIEW

2.1 Surgical site infection global perspective

According to CDC’s National Nosocomial Infection Surveillance system, 38% of all

nosocomial infections in surgical patients were surgical site infections (Goyal et al.,

2015). Surgical site infections were ranked first alongside pneumonia according to a

2011 survey in USA acute care hospitals (Magill et al., 2014). They also constituted

third most common healthcare-associated infection in Australia and Europe according

to literature review (Mitchell et al, 2017; European Centre for Disease Prevention and

Control, 2008).

The rates of surgical site infection worldwide varied greatly, with rates of between 2.6%

-58% recorded (Rosenthal et al., 2013; Donal B O’Connor as part of GlobalSurg

Collaborative, 2018; Apanga et al., 2014; Allegranzi, 2014; Kaur et al., 2017). The

United states of America alone reported an estimated annual SSI incidence ranging

from 160,000 to 300,000 cases, with an estimated SSI annual expenditure of between

$3.5 to $10 billion (Loyola University Health System, 2017).

In England, SSI surveillance for the year 2017/2018 by the Public health of England

reported a cumulative SSI incidence of 8.7% for large bowel surgery between (April

2013 and March 2018) with the data showing a drastic increase in SSI incidence

following large and small bowel surgeries in 2017/18 reaching 8.5% and 6.5%

respectively (Public Health England, 2018). Another surveillance report for the period

January 2003-December 2010 by the Health Protection Scotland (HPS) revealed that

1,883 inpatient SSI cases were resulting from 192,007 procedures. This surveillance

observed an SSI rate of 87.9% among discharged patients following caesarean section

(Health Protection Scotland, 2011).

7

On the other hand, a report for the year 2013-2014 generated by the European Centre

for disease prevention and control on 16 European countries showed that there were

18,364 cases of post-operative wound infection arising from 967,191 surgeries. The

report revealed SSI rates of between 0.6-9.5% per 100 surgeries (European Centre for

Disease Prevention and Control, 2016). A separate review of the burden of six

healthcare-associated infections for the year 2011/12 on the European population

recorded an annual of 2,609,911 cases of healthcare-associated infection with SSI

accounting for most of the cases 799,185 per annum followed by healthcare-associated

urinary tract infection at 777,639 per annum (Cassini et al., 2016).

In Southeast Asia, a meta-analysis of several records on the burden of healthcare-

associated infections (HCAIs) produced a pooled overall HCAI prevalence of 9.0% and

a pooled incidence of 7.8% for SSI (Ling et al., 2015). Whereas research carried out in

west India, recorded an overall prevalence rate of 21.9% for HCAI in the surgical wards

with SSI accounting for 10.9% of the HCAI and an incidence rate of 12.72%

(PatelDisha et al., 2011).

Researchers in South Africa and across Africa reported an SSI rate of 4.60% and

cumulative incidence ranging from 2.5% to 30.9% respectively (Nair et al., 2018; Nejad

et al., 2011). In Kenya, the highest incidence rate (37.7%) of SSI was observed at

Kenyatta National Hospital, while the lowest rate (5%) of SSI was observed at Africa

Inland Church (AIC) Kijabe Hospital following a drastic decline from 9.3% after a

bundle of post-interventions comprising of rationalized antibiotic prophylaxis and

improved operating room activities (Opanga et al., 2017; Ntumba et al., 2015).

8

2.2 The burden of surgical site infections

The burden of SSI in the field of surgery is substantial and it ranges from socio-

economic to physical and psychological effects on the patient and healthcare. These

effects include depression, pain, loneliness, anxiety, immobility due to lack of physical

activity, mortality and the huge debts accumulated by patients after spending extra days

in the hospital may leave some patients in financial hardship (Tanner et al., 2012;

Gelhorn et al., 2018; Dal-Paz et al., 2010; Atkinson et al, 2016). Moreover, an

evaluatory study of the impact of SSI on healthcare in six European countries that is

Germany, France, Britain, Italy, Netherlands, and Spain, revealed that SSI’s were

associated with elevated medical expenses arising from treatment, investigations, and

hospitalization charges (Badia et al., 2017).

2.3 The problem of antibiotic resistance in surgical site infections

The levels of SSIs have increased substantially due to the emergence of multi-drug

resistant pathogens and Methicillin resistant Staphylococcus aureus (Adwan et al.,

2016). This increase in antibiotic resistance among SSI bacteria isolates especially in

India has demonstrated that the situation warrants the attention (Dulange and Thorat,

2015; Bhardwaj et al., 2018).

Furthermore, other studies have also observed widespread drug resistance among SSI

bacteria isolates in the proportions of 82.92% and 65.38% respectively (Mengesha et

al., 2014; Bhatt et al., 2014). In addition, the work of another researcher in Nepal

showed that 64 (66.67%) of the bacterial isolates were multi drug resistant, whilst 26

(27.08%) of the isolates were resistant to one antibiotic and only 6 (6.25%) isolates

were found to be sensitive to all the antibiotics tested (Raza, et al., 2013).

9

Several studies also observed varied resistance with Gram-negative rods and Gram-

positive bacteria isolates against broad spectrum antibiotics such as cephalosporin’s,

chloramphenicol and penicillin’s respectively (Clina et al., 2017; Adegoke et al., 2010).

In Tanzania, a study at Muhimbili National Hospital observed that 63% of the bacteria

isolates were multi-resistant to the antibiotics tested (Manyahi et al., 2014). These

widespread antibiotics resistance rates have left surgeons with fewer antibiotics for SSI

treatment. Therefore this study was carried out in order to develope an antibiogram

profile of the common SSI bacteria pathogens for Kenya.

2.4 Risk factors associated with surgical site infections

The factors contributing to SSIs are variable for each patient, depending on the

associated morbidity. These variables are intrinsic (patient related) and extrinsic factors

(procedure related), Intrinsic variables include modifiable factors such as obesity,

smoking, immunosuppressive disease conditions, and non-modifiable factors for

instance age (Anderson et al., 2014; Triantafyllopoulos et al., 2015). Extrinsic variables

include the type of procedure performed, surgery duration, and hospital environment

(Roy, 2011; Diaz V, 2015).

2.4.1 Intrinsic factors

These are patients’ characteristics and they include the following factors.

2.4.1.1 Cigarette smoking

Nicotine present in cigarette smoke is a vaso-constrictor, this substance can interfere

with the wound healing process by reducing the flow of blood rich in supply to the

surgical bed. The reduced flow of blood to the injured tissues can result in the

impairment of the inflammatory cascade (Sørensen, 2012). These events can result in

10

SSI since the body may not be able to deal with bacterial invasion (Kong et al., 2017;

da Costa et al., 2017; Durand et al., 2013).

2.4.1.2 Gender

Several studies have discovered that gender plays an important role in the development

of surgical site infection among surgical patients (Langelotz et al., 2014; Abdi et al.,

2018; Kikkeri et al., 2014; Takahashi et al., 2018). This factor can be linked to sex

hormones for instance testosterone and estrogen hormones. These hormones can disrupt

the wound healing process by modulating genes responsible for inflammation and re-

epithelization (Engeland et al., 2010; Hardman and Ashcroff, 2008).

2.4.1.3 Age

Recent literature show that the young and the elderly are at a greater risk of acquiring

SSI following surgery (Korol et al., 2013; Amoran et al., 2013; Shah et al., 2017; Chu

et al., 2015). The reasons behind age predisposing surgical patients to infection has not

been fully explored. However, a review of factors affecting wound healing process

among the elderly discovered that altered inflammatory response was responsible for

the cases of wound infections among the aged (Guo and Dipietro, 2010).

2.4.1.4 Immunosuppressive disease conditions

In other instances, immune compromising conditions such as diabetes mellitus and

anemia were discovered as factors linked with a higher risk of SSI. This is because

patients with these conditions have reduced immunity which makes it very hard for the

body to clear the infection at the wound site effectively (Akoko et al., 2012; Novelia et

al., 2017; Gelaw and Abdella, 2018).

11

2.4.1.5 Obesity

On the other hand surgical patients with a higher BMI were seen to be at a greater risk

of acquiring surgical site infection (Thelwall et al., 2015; Naphade and Patole, 2017).

This is due to the high tissue mass in obese patients which lowers tissue vascularity,

increases the complexity of the procedure, and reduces oxygen and antibiotics

permeation to the surgical bed. Hence, all these events can increase SSI susceptibility

(Gupta et al., 2008).

2.4.1.6 Preoperative nasal colonization with Staphylococcus aureus

Recent literature show that Staphylococcus aureus colonizes the anterior nares of about

30% of the human population (Lewis, 2018). Within the hospital setting, this bacteria

has the ability to cause wound infections in immune-compromised surgical patients.

This is because the bacteria can be transferred from the nostril to the patient skin and

later into the wound during skin incision (Savage and Anderson, 2013; Tai et al., 2013;

Inoue et al., 2018).

2.4.1.7 Poor nutritional status

A deficiency of micro and macro-nutrients such as vitamins, proteins, carbohydrates,

iron, zinc and magnesium can have a profound effect on the healing process of the

wound after surgery (Guo and Dipietro, 2010). There is sufficient body of evidence to

suggest that serum albumin levels below 3.5 g/dl can increase the susceptibility of

surgical patients to SSI (Yuwen et al., 2017; Lalhruaizela et al., 2017). This is because

low levels of serum albumin in surgical patients are an indicator of a wide range of

comorbid conditions that can impair patient immunity (Moore et al., 2013).

12

2.4.1.8 Wound classification and ASA score

In other instances, a higher wound class and ASA score together with other parameters

such as suppressed immunity have been shown to increase the risk of surgical patients

developing SSI (Carvalho et al., 2017; Aiken et al., 2013; Olowo-okere et al., 2018).

The two systems are widely used by surgeons to assess the degree of wound bacterial

contamination and the pre-operative physical status of the patient during and prior to

surgery (Zinn and Swofford, 2014; American society of anesthesiologist, 2014).

2.4.2 Extrinsic factors

These are factors which are not part of the patient and include the following factors.

2.4.2.1 Types of procedures

The risk of a patient developing SSI also depends on the type of procedure performed,

with some studies observing higher rates of infection in abdominal surgeries such as

cesarean section (Shrestha et al., 2014; Jasim, 2017). These infection rates can be

ascribed to contamination of the surgical site with enteric rods after an incision has been

made. Moreover, surgical procedures involving the insertion of drains and implants

have been seen to put surgical patients at risk of acquiring SSIs (Yoon et al., 2018;

Oliveira et al., 2018; Ikeanyi et al., 2013). This is due to tissue damage and clot

formation during implant insertion which increases the likelihood of biofilm formation

on the implant. These events may lead to SSI onces the host’s defenses have been

breached (Veerachamy et al., 2014).

2.4.2.2 Longer procedure durations

Additionally, surgical procedures lasting for long hours increases the chances of

exposed tissues being contaminated with environmental bacteria. Moreover, long

surgery durations can lead to tissue desiccation with the likelihood of tissue

13

contamination and may also result in many technical errors as result of fatigue of the

operating team (Cheng et al., 2017). Besides, there is a sufficient body of evidence that

suggests that long operation times contribute to SSI susceptibility (Kasatpibal et al.,

2006; Jeong et al., 2013; Shahane et al., 2012; Ameh et al., 2009).

2.4.2.3 Contaminated hospital surfaces

In addition to surgical procedures, the integrity of the hospital environment can also

determine the outcome of surgery. Contaminated hospital surfaces can harbor

pathogenic micro-organisms that have the potential to cause SSI. These grubby hospital

surfaces are due to poor infection prevention and control programs, with most

pathogens originating from the air, healthcare providers and can also be shed from the

patient skin into the hospital environment (Gelaw et al., 2014; Alfonso-Sanchez et al.,

2017; Ngaroua et al., 2016).

2.4.2.4 Non-sterile equipments

Likewise is the use of surgical instruments contaminated with bacteria from the theater

team. There have been a few documented cases of SSI arising from contaminated

surgical instruments as a result of poor instrument handling and lack of proper

inspection before surgery (Dancer et al., 2012).

2.4.2.5 Hair removal

Hair removal is essential for ease of accessing the operative site, however shaving of

the surgical site using a razor a day before an operation can result in minute skin cuts

that can serve as sites for bacterial build-up and proliferation. The bacteria colonizing

these sites may enter the surgical wound during skin incision and consequently cause

post-operative wound infection (National Collaborating Centre for Women’s and

Children’s Health(UK), 2008; Buteera and Orth, 2008). Besides, clinical trials have

14

shown that hair removal using a razor is associated with a higher risk of SSI

(Faruquzzaman et al., 2012; Tanner et al., 2011).

2.5 Pathogenesis of surgical site infections

The development of a surgical site infection depends on bacterial contamination of a

surgical site. Quantitatively, it has been shown that if a surgical site is contaminated

with >105 bacteria per Gram of tissue, the risk of SSI is markedly increased (Weston et

al., 2016; Greene et al., 2010). However, the dose of contaminating bacteria required

to produce infection may be much lower when foreign material is present at the site

(Damani, 2011).

The probability of wether bacterial contamination can lead to SSI is determined by an

interplay between four factors. These are bacteria inoculum, virulence, adjuvants

effects and host immunity and this can be conceptualized by the following relationship

(Fry, 2013).

Probability of SSI =bacteria inoculum + virulence + adjuvant effects

host immunity

The infection process increases proportionally as the inoculum size and virulence of the

bacteria increases. Local features of the wound like presence of blood (hemoglobin)

and foreign materials like drains potentiate bacterial virulence (Fry, 2013). Most of the

bacteria that cause SSI secrete toxins and possess other invasive structures such as

adhesins that increase their ability to adhere to host cells, penetrate, colonize and

produce damage within the host tissues (Kaye, 2011; Lachiewicz et al., 2015). Some of

the bacterial species can produce an extrapolymeric substance or possess a

polysaccharide capsule, which shields them from both the hosts immune response and

most antimicrobial agents (Crossley et al., 2010).

15

In attempts to clear the invasion, the host’s pro-inflammatory cells releases tumour

necrosis factor- alpha which stimulate neutrophils for bacterial phagocytosis. It also

causes the release of reactive oxygen and acid hydrolases from lysosomal vacuoles

resulting in lipid peroxidation, release of interleukins, evoking inflammatory response

with creation of space containing pus which contain necrotic tissue, neutrophils,

bacteria and proteinaceous fluid with signs of inflammations (rubor, dolor, calor and

tumor) (Sriram, 2016). The micro-environment of purulent collection is relatively

hypoxic and acidotic, thus most cellular and enzymatic functions are inhibited.

Therefore these areas require operative drainage in addition to antibiotic therapy

(Lawrence, 2013).

2.6 Sources of surgical site infections

Pathogens causing surgical site infection can originate from either exogenous or

endogenous sources.

2.6.1 Endogenous sources

Most of the SSIs are due to bacterial contamination of the surgical wounds with

endogenous flora of the host skin and viscera. This contamination occurs after the skin

or hollow viscera has been incised (Gupta, 2006; Newman et al., 2008). These

endogenous flora include skin micro-organisms for instance Staphylococcus aureus and

enteric Gram-negative rods for example E.coli. These pathogens can also originate from

infective sites remote from the surgical site (Lubin et al., 2013; Sarrafzadeh-Rezaei et

al., 2006).

16

2.6.2 Exogenous sources

The sources include the operating room environment, surgical personnel-especially

members of the surgical team, and all instruments brought to the operating room during

an operation (Zhiqing et al., 2018; Dancer et al., 2012; Rello et al., 2007).

Contamination occurs as a result of patient interaction with the surgical team or other

healthcare providers colonized with pathogens and contaminated hospital surfaces with

wounds serving as routes of infection. Exogenous microbiota include Staphylococcus

aureus and Streptococci species (Lee et al., 2013; Singh et al., 2014).

2.7 Pathogen’s causing surgical site infections

Most studies show that the distribution of pathogens isolated from SSIs has not changed

markedly during the last decade. Staphylococcus aureus, Coagulase-negative

staphylococci, Enterococcus species and Escherichia coli remain the most frequently

isolated bacteria. A plethora of evidence indicate that an increasing proportion of SSIs

is due to multidrug-resistant bacteria, Methicillin-resistant Staphylococcus aureus, and

ESBL producers (Seni et al., 2013; Chaudhary et al., 2017; Ramesh and Dharini, 2012;

Naik and Deshpande, 2011; Bhave et al., 2016).

A report from surveillance of National Health Service (NHS) Hospitals in England for

the year 2014/15 discovered that Staphylococcus aureus was the prevalent isolate from

orthopedic and spinal operations accounting for more than 36% of the infections.

However, Coagulase negative staphylococci and enterobacteriaceae were dominant in

patients who had undergone heart bypass and colon surgical procedures respectively.

Staphylococcus aureus also accounted for 13% of SSIs among admitted patients after

a decrease between 2006 and 2007 due to a decline in MRSA (Public Health England,

2015).

17

In a separate report by Health protection agency (England), enterobacteriaceae

accounted for 29% of the total bacteria followed by Staphylococcus aureus at 24%

(349) with MRSA accounting for 4% of all Staphylococci species and 18% of

Staphylococcus aureus. Polymicrobial was reported in procedures involving the gastro-

intestinal 30.3% (314/1,036) and orthopedic 27.6% (162/588) while monomicrobial

was generally high in clean surgeries (Health Protection Agency, 2012).

Additionally, several researchers observed that Staphylococcus aureus was the

predominant SSI bacteria followed by Pseudomonas aeruginosa, E.coli, Klebsiella

species, Citrobacter and Acinetobacter species (Biradar and Roopa, 2015; Suryawanshi

et al., 2014; Subrata, 2016; Shriyan et al., 2010; Shinde and Kulkarni, 2017; Khorvash

et al., 2008). However, a separate project discovered that the most frequent SSI isolate

was Pseudomonas species (42.85%) followed by Klebsiella species (28.5%) (Janugade

et al., 2016).

In Nigeria, India, Switzerland, and Greece, reports by different research workers

reported Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,

Enterococcus, Pseudomonas aeruginosa, and Coagulase negative staphylococci as the

most prevalent isolates in post-operative wound infections (Akinkunmi et al., 2014;

Mundhada and Tenpe, 2015; Amutha et al., 2014; Sawhney et al., 2017; Misteli et al.,

2011; Alexiou et al., 2017). Investigations by other research workers in two separate

countries discovered that SSIs were also caused by anaerobic bacteria such as

Bacteroides fragilis, Clostridium perfringens and atypical pathogens for example

Mycoplasma hominis and Ureaplasma species (Akhi et al., 2015; Barie, 2017).

Evaluations of ward surfaces, operating room environment, non-critical healthcare tools

and bedside surfaces of different hospitals revealed that a lot of the contaminants were

18

Coagulase negative staphylococci, Staphylococcus aureus, Klebsiella species

Pseudomonas species, Aspergillus species, Bacillus species and Methicillin resistant

Staphylococcus aureus (Getachew et al., 2018; Matinyi et al., 2018; Weldegebreal et

al., 2019; Yuen et al., 2015) .

On the other hand, the examination of SSI pathogens in Ethiopia observed an overall

bacterial prevalence of 75.6%, with S.aureus 33.3% and E.coli 14.3% as the most

common bacteria isolates.(Asres et al., 2017). Separate analysis of post-operative

wound infection in Ethiopia isolated 111 pathogenic bacteria from clinical swabs of

which Staphylococci species and Gram-negative enteric rods were the predominant

isolates. This analysis also saw the isolation of Coagulase negative staphylococci 41

(41.8%) as the prevalent isolate followed by Staphylococcus aureus and Pseudomonas

aeruginosa from 75 different sites of the hospital environment. The project recorded

MRSA rates of 9 (34.6%) and 2 (2.0%) from clinical and environmental samples

respectively (Amare et al., 2011).

Similar work in Ethiopia isolated a total of 268 bacteria pathogens from all the

processed specimens. A total of 142 (52.9%) isolates were recovered from medical

devices and air, while the rest of the pathogens were from the health professionals 77

(28.8%) and patients 49 (18.3%) respectively. This analysis identified Staphylococcus

species and Gram-negative rods as causative agents of post-operative wound infection

(Gelaw et al., 2014). In Tanzania and Kenya, the preponderant bacteria isolates from

SSI were Gram-negative enteric rods and Staphylococcus aureus (Mwambete and

Rugemalila, 2015; Okello et al., 2018).

19

2.8 Diagnosis of surgical site infections

When diagnosing surgical site infections, the first step is physical examination of the

patient for signs of infection such as erythema, localized swelling, pain, warmth, and

purulent discharge (Thomas et al., 2016). The second and most important is culture and

sensitivity of pus aerobically or anaerobically to establish the microbiological profile

and antibiogram (Coventry, 2014). Other investigations that may be relevant include

Magnetic Resonance Imaging (MRI) and C-reactive protein levels, Ultrasonography,

Computed tomography scan, sedimentation rates and a complete blood count that

determine whether leukocytosis and neutrophilia are present, especially when the

infection becomes systemic (Kasliwal et al., 2013; Awe and Harrop, 2010).

2.9 Complications of surgical site infections

In most cases, if surgical site infection isn’t managed properly. The infection may

progress from serious to life threatening condition such as, pneumonia due to prolonged

hospital stay, cosmetically unacceptable scars, persistent pain, and movement

restriction (Lyden, 2016; Britt et al., 2012). The patient may also experience

compromised emotional well-being, necrotizing fasciitis, wound dehiscence, metastatic

abscesses, septicemia, prolonged wound healing, organ failure and incisional hernia

(Millar et al., 2011).

2.10 Treatment and Management of surgical site infections

Treatment strategies for SSIs are varied but they involve wound opening with

debridement and drainage to remove all foreign materials, necrotic and infected tissues

(Leaper et al., 2013). In incisional SSIs, the infected wounds are managed by irrigation

with isotonic solutions like saline solutions to remove loose dead tissues and exudates

(Cameron and Cameron, 2013). In addition, there is the use of dair protocol for

treatment of patients who have undergone instrumented spinal surgery. The dair

20

protocol can be implemented successfully within 3 months by performing wide removal

of dead tissues, implant retention, use of antibiotics, and by irrigating the wound with

sterile saline solutions (Manet et al., 2018).

Moreover, the appropriate use of dressings promote wound healing by providing the

wound with a balanced moist healing environment, removes wound exudates, and

protect the wound from harmful germs (Keast and Swanson, 2014). Likewise, is the use

of vacuum-assisted closure (VAC) dressing. In this technique, the wound surface is

exposed to a controlled negative pressure, this removes localized fluid, increases blood

flow, and decreases bacterial load, and stimulates the proliferation of reparative

granulation tissue. This system also assists in the debridement of necrotic tissue and act

as a sterile barrier (Patmo et al., 2014; Karaaslan et al., 2014).

Another treatment option is the use of citric acid, the topical application of citric acid

work against Pseudomonas aeruginosa and Staphylococcus aureus both of which are

SSI pathogens (Nagoba et al., 2011). Additionally, great success has been seen with

the use of electrical stimulation and hyperbaric oxygen therapy in open surgical wounds

(Orsted et al., 2010). For systemic features of infection such as widespread cellulitis,

empiric systemic antibiotics based on sensitivity report should be administered

(Townsend Jr et al., 2012).

21

CHAPTER THREE

MATERIALS AND METHODS

3.1 Study area

The project was carried out at Mama Lucy Hospital. This is a level 5 county government

hospital located in the eastern part of Nairobi. The hospital sits on a 10 acre piece of

land, with its location serving as an excellent catchment area for patients from the whole

of Eastlands and eastern bypass. Since its inception, the facility has reduced the pressure

on Kenyatta National Hospital in that it also serves as a referral facility for patients

from areas within and outside Nairobi. The hospital’s outpatient section serves 800

patients daily and the facility has a bed capacity of 137 beds and an inpatient admission

rate of 131%. This facility offers a wide range of services ranging from casualty and

general outpatient, maternity, laboratory, pharmacy, nutrition, major and minor

surgeries, radiology and orthopedic services.

3.2 Study design

This was a cross-sectional descriptive study.

3.3 Study population

The study included patients of all age group with post-operative wound infections

following general, obstetrics and gynecologic surgeries. All the patients were drawn

from the hospital’s main wards which were surgical wards, maternity wards and

pediatric ward. This research work was done between October 2018 and March 2019.

3.4 Patient recruitment

All the patients who consented and met the inclusion criteria were recruited into the

project. Consent was obtained by signing of the consent forms by the participants. For

children, parents or guardians of legal age would sign the consent forms on their behalf.

22

3.4.1 Inclusion criteria

a) Patients with SSIs and who had consented to participate in the study

b) Patients of all age groups but who had SSIs.

3.4.2 Exclusion criteria

a) Those patients who didn’t develop surgical site infection during the research

period.

b) Those patients who didn’t consent.

3.5 Sample size determination

The appropriate sample size was determined using the Fischer et al., (1998) method:

N =Z2P(1 − P)

d²

Where:

N = sample size required

Z = 1.96 standard error

P = the proportion of antibiotic resistant bacteria isolates from SSIs in Kenya was

unknown (50% value was used)

d= corresponded to margin of error (precision), which was 5 %.

N =1.962𝑥 0.5(1 − 0.5)

0.05² N =

3.8416𝑥0.5𝑥0.5

0.0025

N =0.9604

0.0025 N = 384

The sample size was reduced to 58 using the finite population correction formulae for

proportions. The formulae incorporated the total number of SSI cases (68) observed at

23

Mama Lucy Hospital within a period of 6 months that is between July and December

2018.

n =n0

1 +(n0 − 1)

N

n =384

1 +384 − 1

68 n = 58

Where by (n) was the reduced sample size, (n0) was the original sample size 384 while

(N) was the total population of patients with surgical site infection between July and

December 2018 which was 68 in total. After calculation the sample size obtained was

58 patients and in addition to 68 environmental samples, totaling to 126 samples.

3.6 Sampling technique

The study adopted purposive sampling, with those patients having surgical site

infection characterized by purulent wound discharge recruited into the project. The

patients with suspected surgical site infection were identified by surgeons during the

routine daily ward rounds. The surgeons would then document the clinical signs of

infection in the patient file. Identifying patients with SSI in the maternity wards was a

lot easier because they were kept in one room known as the post-operative day (POD),

while in the rest of the wards the doctor or clinician on duty would assist in the

identification process. Participants were recruited every week and all the patients were

briefed about the research and informed consents obtained prior to their inclusion as

study participants. This included patients with SSI infection in the hospital wards.

Three wards that is surgical, maternity, pediatric and an operating room where patients

and healthcare providers had frequent direct body contact with the surfaces, were

assesed for bacterial contamination. The predefined areas were selected through risk

24

assessment. These areas included the floor, hospital bed handles, operating table,

anaesthetic machine, surgical pack, drip stands, surgical lamp, ward screens, ward

bedside tables, suction machine, sinks and tap handles.

3.7 Sample collection and analysis

3.7.1 Patients pus swabs

Asceptic collection of all the samples was done in the morning during wound dressing

before the wound was cleaned with an antiseptic. To ensure the integrity of the samples,

all the wound surfaces were cleaned first with sterile normal saline to prevent the

introduction of skin normal flora into the pus. During sample collection, all the sterile

swabs were first moistened with sterile normal saline, this was followed by collection

of pus from the deep viable tissues of the wound using moistened sterile cotton wool

swabs by Levine method (rotating the swab over 1 cm² area of viable tissues for 5

seconds) (Cooper, 2010). Two swabs were collected from each patient, one for Gram

stain and the other one for culture. After collection, all the pus swabs were carefully

labelled with patient details and transported to the laboratory of the department of

medical laboratory science Kenyatta University on Stuart transport media within 2

hours of collection.

3.7.2 Wards and operating room surface swabs

The swabbing of the surfaces of the wards and operating room was carried out following

guidelines by the International Organization for Standardization (ISO, 2003). The

collection of the surface swabs was done by stroking the moistened swab in close

parallel sweeps over the defined sampled area while rotating it slowly. For areas such

as the floor which is covered by square tiles. Adequate spacing was taken into

consideration to ensure that every part of the floor was covered. In total 68 surface

swabs were collected for bacterial culture. Once each surface swab had been collected,

25

it was carefully labelled, packaged in a leak proof biohazard bag and transported to the

laboratory of the department of medical laboratory science Kenyatta University on

Stuart transport media within 2 hours of collection. Upon reaching the laboratory all

the specimens were carefully inspected and their details entered in a book.

3.7.3 Microscopy

Smear preparation, staining and examination of the collected pus was done according

to standards prescribed in bacteriology (Cheesbrough, 2006). The first step was

preparation of smears on slides with frosted ends. Each slide was labelled with a patient

number using a lead pencil, the slides were then air dried in a dust free environment,

heat fixed by passing them three times through a flame of spirit lamp. The heat fixed

slides were allowed to cool and stained using Gram staining technique (appendix IV).

After staining, the backs of the slides was wiped using a clean gauze and the slides

placed on a draining rack to air dry. The dried smears were examined microscopically

using the oil immersion objective. Smears of discreet colonies from cultured swabs

were also prepared and stained by Gram stain and observed microscopically.

3.7.4 Culture

Culture of pus and surface swabs was carried out according to the set standards and

procedures in bacteriology (Cheesbrough, 2006). A small amount of the specimen was

applied on the agar surface of both MacConkey and Blood agar (appendix IX and X).

Then using a sterile wire loop, the specimen was spread on the agar surface using the

streaking method (figure 3.1). Each swab was inoculated on a separate plate and after

labelling them, the plates were incubated aerobically at 35-37°C for 18-24 hours. After

incubation, Individual bacteria isolates were identified from their respective plates by

observation of the growth pattern which included checking the form (circular),

elevation (raised, flat or convex), margin (undulate or entire), opacity (translucent,

26

opaque or transparent), hemolysis (beta, alpha or gamma), surface (smooth, dry or

mucoid) and pigmentation (pink, golden yellow or white) of the colonies. Swarming

characteristics of bacteria on blood agar surface was also used in the identification

process (appendix VII). Plates with no growth after 18 hours of incubation were re-

incubated while those with mixed growth were sub-cultured on separate plates until

pure growth of discrete colonies was observed. Reporting of no growth was only done

after the plates were incubated for 48 hours.

Figure 3.1: Streaking method

3.7.5 Biochemical tests

In addition to microscopy and cultural characteristics, various biochemical tests were

carried out in order to identify the isolated bacteria. These tests included catalase test,

coagulase test, urease test, oxidase test, indole, methyl red and voges proskauer tests.

3.7.5.1 Catalase test

This test was done to differentiate catalase producing bacteria such as Staphylococcus

species from the non-catalase producing ones like Streptococcus species. Catalase

enzyme acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water.

sample

27

The test was done by bringing a test organism into contact with a drop of 3% hydrogen

peroxide. The release of oxygen bubbles were indicative of catalase producing bacteria

whilst non-catalase producers were indicated by no bubbles (Apurba and Bhat, 2018).

3.7.5.2 Coagulase test

The test was carried out to identify and differentiate coagulase positive Staphylococcus

aureus from non-coagulase staphylococcus like Staphylococcus epidermidis (appendix

VIII). In this test, a drop of distilled water was placed on each end of the slide. Carefully

selected discrete colonies of the test organism were emulsified with the drops of water

on each end of the slide, with one suspension acting as a control. The formation of

clumps 10 seconds after the addition of a loopful of plasma to one of the suspension

demonstrated that the test was positive. All negative slides tests were subjected to a test

tube coagulase test (Cheesbrough, 2006).

3.7.5.3 Urease test

This test was performed in order to identify those bacteria with the ability to hydrolyze

urea using urease enzyme. In this test, the test organism was inoculated in a test tube

medium containing urea and phenol red indicator. The other uninoculated tube acted as

a control. After 24-48 hours of incubation at 37°C, both tubes were examined for colour

change. The change in colour of phenol red from light orange to pink meant that the

test was positive, whilst the lack of phenol red colour change indicated that the test was

negative (appendix V). Some of urease positive bacteria include proteus species and

Klebsiella pneumoniae (Hemraj et al., 2013).

28

3.7.5.4 Oxidase test

This was carried out by picking a colony of the test organism and smearing it over a

filter paper impregnated with oxidase reagent. The formation of purple colour indicated

a positive test for oxidase producing bacteria like Pseudomonas aeruginosa, whilst no

colour change demonstrated that the test was negative for bacteria such as Escherichia

coli. (Cheesbrough, 2006).

3.7.5.5 Indole test

Indole test was carried out in order to identify those bacteria with the ability to

breakdown tryptophan into indole such as Escherichia coli. In this test, the test

organism was inoculated in tryptone broth and the tubes incubated at 37°C for 48 hours.

After incubation, the indole was identified by adding 0.5ml of kovac’s reagent to the

inoculated tubes. The formation of a red ring on the surface of tryptone after gentle

shaking demonstrated that the test was positive, while no red layer meant that the test

was negative (appendix V) (Cheesbrough, 2006).

3.7.5.6 Methyl red and Voges proskauer tests

Methyl red and voges proskauer tests were used in the identification of

enterobacteriaceae such as Escherichia coli which is methyl red positive and voges

proskauer negative. Both tests were carried out by inoculation of test organism in

methyl red-voges proskauer broth. After 24 hours of incubation at 35°C, 1ml of the

broth was transferred to a clean tube and the remaining broth re-incubated for another

24 hours (Hemraj et al., 2013).

The 1ml broth that was transferred to a clean test tube was later used for voges-

proskauer test. To the 1ml broth, 0.6ml of 5% alpha-naphthol was added followed by

0.2ml of 40% potassium hydroxide. Following gentle shaking, the formation of a red-

29

pink colour on the surface of the medium after 10 minutes demonstrated that the test

was positive. Whereas the development of a yellow colour on the surface of medium

demonstrated that the test was negative. Examples of voges proskauer positive bacteria

are enterobacter and Klebsiella species.

In methyl red test, 2.5ml of the 48 hour broth was transferred to a clean tube. The

formation of a red colour on the surface of the medium after the addition of 5 drops of

methyl red, demonstrated that the test was positive. Negative methyl red tests were

demonstrated by the development of a yellow colour on the surface of the medium

(Hemraj et al., 2013).

3.7.5.7 Citrate test

Citrate test was performed in order to identify those bacteria with the ability to utilize

citrate as their sole carbon source. This test was performed by streaking the slope and

stabbing the butt of Simmons citrate agar with the test organism using a sterile straight

wire. The tubes were later incubated at 37°C for 48 hours and the change in colour of

the agar from green to blue indicated that the test was positive (appendix V). The most

notable bacteria that are citrate positive and citrate negative are Klebsiella pneumoniae

and Escherichia coli respectively (Cheesbrough, 2006).

3.7.6 Antibiotic susceptibility testing

Antibiotic sensitivity tests were performed on the bacteria isolates by Kirby Bauer

antibiotic testing method according to the Clinical and Laboratory Standards Institute’s

guidelines (appendix XI) (Cavalieri et al., 2005). In this technique, 3-5 pure colonies

of the test organism were emulsified in 3-4 ml of sterile physiological saline and the

suspension adjusted to match that of 0.5 McFarland turbidity standard. A sterile swab

was dipped in the tube with the saline suspension and pressed against the side of the

30

tube above the suspension to remove the excess fluid, this was followed by uniform

coating of the Mueller Hinton surface with the test organism in three directions and in

each instance rotating the plate at an angle of 60° to ensure even coating. The plate’s

surfaces were given 3 minutes to dry with their lids in place. Then using sterile forceps,

the antibiotic discs were placed uniformly on the inoculated agar surface. These plates

were inverted and incubated aerobically at 35°C for 16-24 hours and after incubation

the test plates and control were examined for confluent growth. When using Oxacillin,

all the plates were incubated for a full 24 hrs at 35°C. The diameters of the resultant

zones of inhibition on each plate were measured in millimeters and interpreted as either

sensitive, intermediately sensitive or resistant based on the criteria and breakpoints set

by the CLSI, EUCAST and BSAC. When interpreting Oxacillin resistance in

Staphylococcus species, all Oxacillin resistant Staphylococcus aureus were taken to be

methicillin resistant Staphylococcus aureus (CLSI, 2016; European Committee on

Antimicrobial Susceptibility Testing, 2019; BSAC, 2013).The study also included

Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 as reference

strains (CLSI, 2016).

3.8 Data collection tool

The questionnaire captured the demographics, admission details and clinical

characteristics of the participants (appendix II). These questionnaires were

administered by the principal investigator in both English and Swahili languages.

3.9 Ethical consideration

Approval for study was sought from the Kenyatta University Ethics Review Committee

(KUERC) (appendix III). Authority to collect data was acquired from the research

committee of Mama Lucy Kibaki Hospital (appendix XII). All the participants were

briefed about the study and informed consent obtained from each of the participant

31

(appendix I). For minors, parents or guardians would consent on behalf of the child.

The research was voluntary and those patients who didn’t take part were given the same

treatment and care as those who took part. Pus was collected using a soft-sterile

moistened swab minimizing any risk of contamination of the wound and subsequent

pain. Details of each participant were extracted from the hospital records using

serialized questionnaire, the same serial number on each questionnaire was maintained

during coding therefore ensuring the anonymity of the participant details. Patient

records were kept safe in lockable cabinets and password-finger print protected laptop

accessed only by the principal investigator. All the study participants received treatment

that was guided by sound bacteriology reports coupled by regular wound care (dressing)

by the nurse on duty or intern and the attending physician.

3.10 Data analysis

The antibiogram report and data extracted from the questionnaire containing details of

each patient were used to determine descriptive statistics like mean, range and

percentages with the help of a Statistical Package for Social Sciences (SPSS) version

20. The association between gender and the different types of bacteria was analyzed

using Chi-square (p<0.05). Results generated for each patient were presented in form

of tables and charts.

32

CHAPTER FOUR

RESULTS

4.1 Patients demographics

In the present study, a total of 58 patients who had surgical site infection within the

research period were enrolled. Out of these 19 (32.8%) were males whilst 39 (67.2%)

were females, with pediatric patients accounting for 3 (5.2%) of the total cases. The

disease incidence was high among patients of the age group 18-45 years, whilst low

cases of SSI were observed in patients below 18 years (figure 4.1). The youngest

participant was 7 years old while the eldest was 61 years and the participants had a

mean age of 31.12 years.

Figure 4.1: Patients age distribution

4.2 The prevalence of bacteria isolated from surgical site infections

There were a total of 78 bacteria isolated from culture positive swabs. The prevalence

of SSI isolates was as follows; Staphylococcus aureus 28.2% (n=22) was the prevalent

bacteria followed by E.coli 15.4% (n=12), Acinetobacter species 14.1% (n=11),

Pseudomonas aeruginosa 9.0% (n=7), Enterobacter species 9.0% (n=7), Bacillus

3

47

80

5

10

15

20

25

30

35

40

45

50

below 18 18-45 above 45

Count

Age (years)

33

species 9.0% (n=7), Coagulase negative staphylococci 5.1% (n=4), Proteus species

5.1% (n=4), Klebsiella pneumoniae 2.6% (n=2), Morganella morganii 1.3% (n=1) and

Citrobacter freundii 1.3% (n=1) (figure 4.2).

Figure 4.2: The prevalence of bacteria isolated from patients with surgical site

infection at Mama Lucy Hospital, Staphylococcus aureus was the prevalent SSI

bacteria isolate at the hospital.

The majority of the SSI bacteria were Gram-negative rods 57.7% (45) with Gram-

positive bacteria accounting for the rest of the isolates 42.3% (33). The proportion of

SSI isolates in female 73.1% (57) was more compared to male 26.9% (21) but the

difference was not statistically significant (x² = 2.221, p<0.136) because the p-value

was higher than the set alpha level of significance of 0.05.

4.3 The antibiotic sensitivity pattern of surgical site infection bacteria

Gram-positive bacteria were sensitive to Chloramphenicol 90.9% (n=30/33),

Vancomycin 87.9% (n=29/33), Doxycycline 75.8% (n=25/33) and Ciprofloxacin

69.7% (n=23/33). Whereas, Gram-negative rods had sensitivity to Chloramphenicol

0

5

10

15

20

25

30

Pre

val

ence

( i

n p

erce

nta

ges

)

Types of bacteria isolates

34

53.3% (n=24/45) and Amikacin 50% (n=10/20). The least sensitivity of Gram-positive

and Gram-negative bacteria was observed with Ampicillin/Cloxacillin 3.0% (n=1/33)

and Amoxycillin 3.7% (n=1/27) drugs respectively (Tables 4.1 and 4.2).

Table 4.1: The antibiotic sensitivity pattern of Gram-positive bacteria from

patients with surgical sites infections at Mama Lucy Hospital.

Staphylococcus

aureus

(N=22)

Coagulase

negative

staphylococci

(N=4)

Bacillus

species

(N=7)

Total

(N=33)

Antibiotics tested S I R S I R S I R S I R

Vancomycin 30ug 20 0 2 3 1 0 6 0 1 29 1 3

Ciprofloxacin 30ug 17 2 3 4 0 0 2 3 2 23 5 5

Chloramphenicol

50ug

21 0 1 4 0 0 5 1 1 30 1 2

Doxycycline 30ug 19 2 1 2 1 1 4 1 2 25 4 4

Ampicillin/

Cloxacillin10ug

1 0 21 0 0 4 0 0 7 1 0 32

Azithromycin 15ug 11 1 10 1 0 3 0 1 6 12 2 19

Oxacillin 1ug 6 3 13 0 0 4 0 0 0 6 3 17

Key: S- Sensitive I- Intermediately Sensitive R- Resistant

35

Table 4.2: The antibiotic sensitivity pattern of Gram-negative bacteria from

patients with surgical sites infections at Mama Lucy Hospital.

Bacteria susceptibility CPM

30ug

AK

30ug

CTR

30ug

CIP

30ug

C

50ug

DO

30ug

COT

25ug

GEN

10ug

AMX

30ug

Morganella

morganii

N=1 S 0 0 0 0 1 0 0 1 0

I 0 0 0 1 0 0 0 0 0

R 0 0 0 0 0 1 1 0 1

Proteus

species

N=4 S 0 0 0 2 2 0 2 4 0

I 0 0 0 2 2 0 0 0 0

R 0 0 0 0 0 4 2 0 4

Klebsiella

pneumoniae

N=2 S 0 0 0 0 1 0 0 0 0

I 0 0 0 1 0 1 0 0 0

R 2 2 0 1 1 1 2 2 2

E.coli N=12 S 2 0 2 3 5 4 0 5 0

I 0 0 0 2 0 0 0 1 1

R 10 0 10 7 7 8 12 6 11

Pseudomonas

aeruginosa

N=7 S 2 5 0 5 4 2 0 4 0

I 0 0 0 0 1 0 0 0 0

R 5 2 0 2 2 5 0 3 0

Enterobacter

species

N=7 S 0 0 0 5 6 3 3 3 0

I 0 0 0 1 0 0 0 1 0

R 0 0 0 1 1 4 4 3 7

Acinetobacter

species

N=11 S 0 5 0 5 4 3 0 4 0

I 0 0 0 3 5 1 0 0 0

R 11 6 0 3 2 7 0 7 0

Citrobacter

freundii

N=1 S 0 0 0 0 1 1 0 1 0

I 0 0 0 0 0 0 0 0 0

R 0 0 0 1 0 0 1 0 1

Total N=45 S 4 10 2 20 24 13 5 22 0

I 0 0 0 10 8 2 0 2 1

R 28 10 10 15 13 30 22 21 26

CPM- Cefepime AK-Amikacin CTR-Ceftriaxone CIP-Ciprofloxacin

C-Chloramphenicol DO-Doxycycline GEN-Gentamicin AMX-Amoxycillin

S- Sensitive I- Intermediately Sensitive R- Resistant

36

4.4 The resistance pattern of surgical site infection bacteria

The results presented in tables 4.3 and 4.4 show that the most resistant SSI bacteria

isolates were Gram-negative rods with Klebsiella pneumoniae and E.coli recording

resistance rates of 81.3% and 74% respectively. Among the Gram-positive bacteria,

MRSA accounted for 65.4% (n=17/26) of all Staphylococci species, 59.1% (n=13/22)

of the total Staphylococcus aureus and 100% (n=4/4) of all Coagulase negative

staphylococci (CoNS). Overall the observed resistance rate across all isolates was

49.4%.

Table 4.3: The resistance rate of Gram-positive bacteria isolated from surgical

site infections against the drugs tested.

VA

30ug

CIP

30ug

C

50ug

DO

30ug

AX

10ug

AZM

15ug

OX

1ug Total

Bacteria resistance (%)

Staphylococcus aureus 9.1 13.6 4.5 4.5 95.5 45.5 59.1 33.1

CoNS 0.0 0.0 0.0 25.0 100.0 75.0 100.0 42.9

Bacillus species 14.3 28.6 14.3 28.6 100.0 85.7 - 45.2

Total 9.1 15.2 6.1 12.1 97.0 57.6 65.4 36.6

VA-Vancomycin, CIP-Ciprofloxacin, C-Chloramphenicol, DO-Doxycycline, AX-

Ampicillin/cloxacillin, AZM-Azithromycin, OX-Oxacillin, CoNS- Coagulase

negative staphylococci.

37

Table 4.4: The resistance rate of Gram-negative bacteria isolated from surgical

site infections against the drugs tested.

CPM

30ug

AK

30ug

CTR

30ug

CIP

30ug

C

50ug

DO

30ug

COT

25ug

GEN

10ug

AMX

30ug

Total

resistance

Bacteria

(%) Morganella

morganii

- - - 0.0 0.0 100.0 100.0 0.0 100.0 50.0

Proteus

species

- - - 0.0 0.0 100.0 50.0 0.0 100.0 41.7

Klebsiella

pneumoniae

100.0 100.0 - 50.0 50.0 50.0 100.0 100.0 100.0 81.3

E.coli 83.3 - 83.3 58.3 58.3 66.7 100.0 50.0 91.7 74.0

Pseudomonas

aeruginosa

71.4 28.6 - 28.6 28.6 71.4 - 42.9 - 45.2

Enterobacter

species

- - - 14.3 14.3 57.1 57.1 42.9 100.0 47.6

Acinetobacter

species

100.0 54.5 - 27.3 18.2 63.6 - 63.6 - 54.5

Citrobacter

freundii

- - - 100.0 0.0 0.0 100.0 0.0 100.0 50.0

Total

resistance

87.5 50.0 83.3 33.3 28.9 66.7 81.5 46.7 96.3 58.7

CIP- Ciprofloxacin, GEN- Gentamicin, AMX- Amoxicillin, C-Chloramphenicol, DO-

Doxycycline, COT-Cotrimoxazole, CTR- Ceftriaxone, CPM-Cefepime, AK-

Amikacin.

4.5 The prevalence of bacteria contaminating the wards and operating room

A total of 59 bacteria were isolated from culture positive surface swabs, with

Staphylococcus aureus 55.9% (n=33) as the predominant bacteria followed by Bacillus

species 30.5% (n=18), Citrobacter species 3.4% (n=2), Enterobacter species 3.4%

(n=2), Pseudomonas aeruginosa 1.7% (n=1), Acinetobacter species 1.7% (n=1), E.coli

1.7% (n=1) and Coagulase negative staphylococcus 1.7% (n=1). Table 4.5 illustrates

the areas where swabbing was carried out, the prevalence and the types of bacteria

isolated.

38

Table 4.5: The prevalence of bacteria contaminating the different sections of

Mama Lucy Hospital.

Surgical

wards

Bacteria N (%)

Pediatric

ward

N (%)

Maternity

wards

N (%)

Operating

room

N (%)

Total

N (%)

Staphylococcus

aureus

9(56.3) 4(50.0) 16(55.2) 4(66.7) 33(55.9)

Bacillus species 6(37.5) 2(25.0) 8(27.6) 2(33.3) 18(30.5)

Coagulase negative

staphylococcus

0(0.0) 0(0.0) 1(3.4) 0(0.0) 1(1.7)

Acinetobacter

species

0(0.0) 0(0.0) 1(3.4) 0(0.0) 1(1.7)

Enterobacter

species

1(6.3) 0(0.0) 1(3.4) 0(0.0) 2(3.4)

Citrobacter species 0(0.0) 1(12.5) 1(3.4) 0(0.0) 2(3.4)

Pseudomonas

aeruginosa

0(0.0) 1(12.5) 0(0.0) 0(0.0) 1(1.7)

E.coli 0(0.0) 0(0.0) 1(3.4) 0(0.0) 1(1.7)

Total no of isolates 16(27.1) 8(13.6) 29(49.2) 6(10.2) 59(100)

Key: N- represents the number of bacteria isolated, (%) - represent the percentages

4.6 The antibiotic sensitivity pattern of bacteria from the wards and operating

room

Majority of the isolates were resistant to Ampicillin/Cloxacillin at 96.6% (n=57/59).

However, most of them were sensitive to Ciprofloxacin 86.4% (n=51/59), Doxycycline

88.1% (n=52/59), Chloramphenicol 93.2% (n=55/59) and Vancomycin 100%

(n=52/52). MRSA accounted for 61.8% (n=21/34) of the total Staphylococci species

(Table 4.6).

39

Table 4.6: The antibiotic sensitivity pattern of bacteria recovered from the wards

and operating room of Mama Lucy Hospital.

Bacteria

susceptibility

VA

30ug

CIP

30ug

C

50ug

DO

30ug

AX

10ug

AZM

15ug

OX

1ug

CTR

30ug

CPM

30ug Total

Staphylococcus

aureus

S 33 29 30 29 2 14 11 - - 148

I 0 2 0 1 0 3 2 - - 8

R 0 2 3 3 31 16 20 - - 75

Bacillus

species

S 18 18 18 18 0 16 - - - 88

I 0 0 0 0 0 2 - - - 2

R 0 0 0 0 18 0 - - - 18

CoNS S 1 1 1 1 0 1 0 - - 5

R 0 0 0 0 1 0 1 - - 2

Enterobacter

species

S - 1 1 1 0 - - 0 0 3

I - 1 1 1 0 - - 0 0 3

R - 0 0 0 2 - - 2 2 6

Citrobacter

species

S - 0 2 2 0 - - 0 0 4

I - 1 0 0 0 - - 0 1 2

R - 1 0 0 2 - - 2 1 6

Acinetobacter

species

S - 1 1 0 0 - - 0 1 3

I - 0 0 0 0 - - 1 0 1

R - 0 0 1 1 - - 0 0 2

Pseudomonas

aeruginosa

S - 0 1 0 0 - - 1 1 3

I - 1 0 1 0 - - 0 0 2

R - 0 0 0 1 - - 0 0 1

E.coli S - 1 1 1 0 - - 1 1 5

R - 0 0 0 1 - - 0 0 1

Total

Total bacteria

S 52 51 55 52 2 31 11 2 3 259

I 0 5 1 3 0 5 2 1 1 18

R 0 3 3 4 57 16 21 4 3 111

52 59 59 59 59 52 34 7 7 388

CIP-Ciprofloxacin, AX- Ampicillin/Cloxacillin, AZM-Azithromycin, OX-Oxacillin,

C-Chloramphenicol, VA-Vancomycin, DO-Doxycycline, CPM-Cefepime, CTR-

Ceftriaxone, CoNS- Coagulase negative staphylococcus

40

CHAPTER FIVE

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

5.1 Discussion

5.1.1 The prevalence of bacteria isolated from surgical sites infections

In present study, the most prevalent SSI bacteria was Staphylococcus aureus 28.2%

(n=22) followed by E.coli 15.4% (n=12). These two bacteria that is Staphylococcus

aureus and E.coli are mostly skin and gastro-intestinal tract flora respectively.

Therefore their high isolation rate from post-operative wounds at the current set-up was

attributed to endogenous contamination of the exposed tissues with skin and gastro-

intestinal tract flora during surgery.

The high prevalence of Staphylococcus aureus observed by the current study was

analogous with findings from other research works (Moses, 2016; Banashankari et al.,

2014; Bastola et al., 2017; Kurhade et al., 2015; Das et al., 2015). However, the

findings by this project were in contrast with several studies which identified E.coli

(25.5%), Acinetobacter species (32.03%), Pseudomonas aeruginosa and Klebsiella

pneumoniae as the predominant SSI bacteria isolates (Nwankwo et al., 2014; Devjani

et al., 2013; Kokate et al., 2017; Patel et al., 2019; Billoro et al., 2019). This difference

was attributed partly on the procedures performed by the hospital, for example surgical

site infection arising following procedures involving the hollow viscera or the skin

could be due to enteric rods or skin commensals like Staphylococcus species.

This project also isolated Bacillus species from patient pus swabs, which proved that

Bacillus species especially Bacillus cereus should not be dismissed as contaminants of

clinical samples. This agrees with findings by different researchers who were able to

isolate Bacillus species from post-operative wounds (Ako-nai et al., 2013; Ebert et al.,

2019).

41

The majority of the SSI bacteria isolates were Gram-negative rods 57.7% (45) with

Gram-positive bacteria accounting for 42.3% (33) of the total isolates. These results

were linked to antibiotic resistance, because majority of the Gram-negative bacteria

isolates were more resistant compared to Gram-positive isolates. These findings were

congruous with observations made by other research works which observed the

predominance of Gram-negative bacteria over Gram-positive bacteria 56.5% versus

43.5% and 85 versus 27 (Tuon et al., 2018; Garg and Singh, 2017). However, the results

were inconsistence with the work of two researchers who observed that Gram-positives

were more compared to Gram-negatives (Khyati et al., 2014; Rolston et al., 2014). The

difference in the types of bacteria isolated from the current setting to those obtained by

the other researchers is because different facilities have different bacteria to which

patients are at risk of acquiring SSI during a given period in time (Gelaw et al., 2014;

Kokate et al., 2017).

5.1.2 The prevalence of bacteria contaminating the wards and operating room

The analysis of the wards and operating room revealed Staphylococcus aureus 55.9%

(n=33) as the preponderant contaminant followed by Bacillus species 30.5% (n=18),

Citrobacter species 3.4% (n=2), Enterobacter species 3.4% (n=2), Pseudomonas

aeruginosa 1.7% (n=1), Acinetobacter species 1.7% (n=1), E.coli 1.7% (n=1) and

Coagulase negative staphylococcus 1.7% (n=1). These results were similar to those

obtained by other research works in that Staphylococcus species, Gram-positive and

Gram-negative rods were the most common isolates (Pradhan and Shrestha, 2012; Saka

et al., 2016; Atata et al., 2010). These bacteria come from either the air, patients, visitors

or even health care workers. Consequently, their presence in these environments can be

explained by their remarkable ability to survive for long on poorly cleaned dry surfaces

(Kramer et al., 2006).

42

In the analysis of the wards and operating room, the proportion of Gram-positive

bacteria isolates (n=52) exceeded that of Gram-negative bacteria (n=7). This results

were in harmony to those of a similar study done in Ethiopia (Gelaw et al., 2014). Since

Gram-positive bacteria can survive for months on dry surfaces, their presence in the

two environments can be ascribed to inadequate cleaning of these surfaces (Kramer et

al., 2006).

Majority of the bacteria isolates were from the maternity wards 49.2% followed by

surgical ward 27.1%, paediatric ward 13.6%, and operating room 10.2% and in all these

areas Staphylococcus aureus was the main contaminant. Staphylococcus aureus was

also seen in 42% of the bacteria that were isolated from the floors of surgical wards of

a hospital in Nigeria (Olowo-okere and Babandina, 2018). These results were contrary

to those from a study of the healthcare environment in Morocco that found

Enterobacteria (31.6%) as the main contaminant (Chaoui et al., 2019). The variation in

the isolates recorded could be ascribed to several factors, for instance, the level of

shedding by patients, culturability of organisms, the difficulty of cleaning a particular

area and sampling methodology (Otter et al., 2013).

5.1.3 The antibiotic sensitivity pattern of bacteria isolated from surgical site

infection

Gram-negative isolates were resistant to Cefepime, Ceftriaxone, Cotrimoxazole, and

Doxycycline. However, relative success was observed with Chloramphenicol and

Amikacin. In another instance, Gram-negative bacteria showed resistance to

Cefuroxime (18.5%) and sensitivity to Lomefloxacin (70.3%) and Netillin (61.1%).

The same research noted that Gram-positive bacteria were resistant to Ciprofloxacin

(52.5%) but sensitive to Ampicillin+Sulbactam (87.5%) and Linezolid (85%) (Insan et

al., 2013). The variation in sensitivity of Gram-positive bacteria to Ciprofloxacin

43

between the present and aforementioned study may be attributed to bacteria mutation

which led to bacteria isolates becoming less sensitive to these antibiotics (eLife, 2017).

This project discovered that a majority of the SSI isolates were highly resistant to

Ampicillin and Amoxycillin proving that the two drugs were no longer very effective

in the treatment of surgical site infection at the hospital. A similar trend of wide spread

resistance had been reported by a number of studies including (Al-Awaysheh, 2018;

Kahsay et al., 2014). However, the relative success of Ampicillin and Amoxycillin has

been documented in India (Gangania et al., 2015). Although the two drugs performed

relatively well. The sensitivity of SSI bacteria isolates to these drugs was actually low,

meaning that bacteria have developed a very high resistance against the two drugs.

The current research discovered that the most resistant SSI bacteria isolates were Gram-

negative rods, with Klebsiella pneumoniae and E.coli recording resistance rates of

81.2% and 74% respectively. This observation was comparable with the work of a

separate worker who reported a resistance rate of 75% among Gram-negative rods

(Dessie et al., 2016). These high resistance rates recorded by this work can be attributed

to the indiscriminate use of antibiotics at the hospital which encouraged bacteria to

evolve into highly resistant pathogens. The facility also lacked mechanisms in place to

identify ESBL producers like Klebsiella pneumoniae and E.coli which may prove to be

difficult to treat for the clinicians.

This study recorded a Methicillin resistant Staphylococcus aureus rate of 65.4% which

was high compared to the rate of 8.6% reported in India by (Bhave et al., 2016).

Nevertheless the results by this work were similar to a study conducted in Uganda that

reported a MRSA rate of 65.9% (George et al., 2018). These high levels of MRSA

recorded in the current set-up may be attributed to the lack of mechanisms for MRSA

44

active surveillance. Moreover, the current setting lacked a sound diagnostic

microbiology laboratory for bacteria culture and relied mainly on empiric therapy. This

lack of antibiogram profiles and poor antibiotic stewardship, may have contributed to

the emergence of bacteria resistance especially with Methicillin resistant

Staphylococcus aureus and other bacteria isolates.

5.1.4 The antibiotic sensitivity pattern of bacteria from the wards and

operating room

Gram-positive bacteria had 100% sensitivity to Vancomycin. Whereas both Gram-

positive and Gram-negative bacteria showed sensitivity to Chloramphenicol 93.2%,

Doxycycline 88.1% and Ciprofloxacin 86.4%. The resistance of all isolates to

Ampicillin/cloxacillin 96.6% was generally high. These findings were in harmony with

a study done in Uganda, of which environmental bacteria isolates were 100% and 80%

susceptible to Vancomycin and Ciprofloxacin respectively (Sserwadda et al., 2018).

The two studies showed that environmental bacteria were more sensitive to the drugs

tested against them. However, not all isolates were susceptible to these drugs, as a result

more work is needed to identify population of resistant bacteria within the hospital

environment.

The MRSA rate of 61.8% (n=21/34) for all staphylococci species observed in the

current set-up was different to the work of another research worker who recorded a

MRSA rate of 73.7% with Staphylococcus aureus and 11.4% with Coagulase negative

staphylococcus (Worku et al., 2018). These high levels of MRSA in the current setting

may be attributed to the failure by the hospital to institute mechanisms for active

surveillance of MRSA, which meant that surgical patients were at risk of acquiring

MRSA infection.

45

5.2 Conclusions

i. Among SSI bacteria isolates, Staphylococcus aureus was identified as the

leading cause of SSI among surgical patients attending Mama Lucy Hospital.

ii. In the wards and operating room of Mama Lucy Hospital, the most prevalent

bacteria contaminant was Staphylococcus aureus.

iii. All SSI bacteria isolates were sensitive to Chloramphenicol, but a majority of

the Gram-positive and Gram-negative SSI bacteria were resistant to Ampicillin

and Amoxycillin respectively.

iv. Almost all environmental bacteria isolates were resistant to Ampicillin but had

sensitivity to Vancomycin, Chloramphenicol, Ciprofloxacin and Doxycycline.

5.3 Recommendations

i. The facility needs to identify the most frequent bacteria isolates associated with

SSI together with their antibiograms.

ii. The hospital needs to monitor bacteria isolates that frequently contaminate the

wards and operating room of Mama Lucy Hospital.

iii. The facility should consider using Chloramphenicol in the treatment of surgical

site infections at the hospital.

5.4 Recommendations for further research

i. Studies should be done on Molecular characteristics of Methicillin resistant

Staphylococcus aureus in surgical site infections.

ii. There should be an elaborate analysis of extended spectrum beta-lactamase

producers (Gram-negative enterics) and constitutive or inducible Clindamycin

resistance in Staphylococcus aureus.

46

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APPENDICES

Appendix I-Consent form

Study title: Antibiotic susceptibility patterns of bacteria isolated from wards, operating

room and post-operative wound infections among patients attending Mama Lucy

Hospital, Kenya.

Principal investigator: Amulioto Johnstone Auna, Bsc. (MLS), Post-graduate Student

Kenyatta University.

Co-investigators: Dr. Margaret Muturi (PHD), Dr. Scholastica Mathenge (PHD)

Aim of the study: To determine the prevalence and antibiotic susceptibility patterns of

bacteria isolated from wards, operating room and surgical sites infections among

patients attending Mama Lucy Hospital.

Procedure: Participation in this study will require that I ask you a few questions before

obtaining a swab from your wound area using a sterile moistened cotton swab for

testing. But please remember that this study is voluntary and your decision to refuse or

take part in the study will not in any way change the care and treatment you will receive

from this hospital today or in future.

Risks and benefits: The study involves collection of wound pus using a painless sterile

moistened cotton swab, although you might experience minimal discomfort during

maneuvering of the skin areas, I guarantee you that it is safe and there will be no harm.

The findings generated from positive samples will help the clinician to administer

appropriate antibiotics for the treatment of your infection.

Cost: This study is free to all patients who are willing to participate, no fee is charged

for participation.

65

Confidentiality: Participants information will be collected on a serialized

questionnaire and the same serial number will be used in coding and this will ensure

that your details remain anonymous. Therefore at no point in time will the name of the

participant appear anywhere in the study. The participants’ records will also be kept in

secure lockable cabinets only accessible by the principal investigator.

Participant:

I have read and understood fully the contents of this form. My questions have been

answered by the investigator. I also understand that the study is voluntary and whether

I decide to participate in the study or not, will not in any way affect my care and

treatment at this hospital. I_______________________________voluntarily agree to

take part in the study mentioned above.

Participant’s Name________________________________

Signature____________ Date__________

The investigator: I confirm that I have answered all your questions regarding this study

and that you have voluntarily consented to participate in the study

Investigator’s name __________________________________

Signature__________________________ Date__________

If you have any queries about this study, feel free to contact the principal investigator

Amulioto Johnstone Auna, P.o box 43844-00100, Nairobi, Tel 0738815499 or Dr.

Margret Muturi, Kenyatta University, Tel 0722758523. Concerns about your rights as

participants have been addressed by Kenyatta University Ethics Review Committee.

66

Appendix II-Questionnaire

Serial number___________

Demographics

Patient name_______________________ Sex________________________

Age________________________ Patient number_______________

Clinical history

Date of admission______________ Date of discharge____________

Length of hospital stay____________

67

Appendix III- KUERC approval letter

68

69

Appendix IV- Gram staining technique

Principle

Gram staining technique is used for differentiating bacteria into two groups which are

Gram-negative and Gram-positive based on the physical and chemical properties of

their cell walls. Gram-positive bacteria have a thick cell wall which can resist

decolourization therefore they retain the colour of the initial stain which is crystal violet.

Whereas Gram-negative bacteria appear pink in colour because of their thin cell wall

which is easily washed away by a slight alcohol wash permitting the escape of the initial

stain and allowing the cell to take the colour of the counterstain.

Procedure

i. The heat fixed smears were flooded with crystal violet stain for 1 minute

ii. The excess stain washed off with clean water

iii. The slides were then covered with lugol’s iodine to fix the stain for 1 minute

iv. The excess iodine washed off with clean water

v. Followed by rapid decolourization using acetone until the acetone running from

the slides was clear.

vi. Washed off the acetone with clean water and counterstained the slides with

neutral red stain for 2 minutes.

vii. Washed off the excess counter stain with clean water, wiped the backs of the

slides with a clean gauze and left the slides to air dry on a draining rack.

viii. Applied immersion oil and observed the slides microscopically x100

Results interpretation

Gram-positive bacteria ……… appear purple example Staphylococcus aureus

Gram-negative bacteria………. appear pink example Pseudomonas aeruginosa

70

Appendix V- Biochemical tests

Indole test

Citrate test

71

Urease test

72

Appendix VI- Publication abstract

Vol. 14(8), pp. 420-425, August, 2020

DOI: 10.5897/AJMR2020.9357

Article Number: EC957D864629

ISSN: 1996-0808

Copyright ©2020

Author(s) retain the copyright of this article

http://www.academicjournals.org/AJMR

African Journal of Microbiology Research

Antibiotic susceptibility patterns of bacteria isolates from post-operative wound

infections among patients attending Mama Lucy Kibaki Hospital, Kenya

Johnstone Amulioto1*, Margaret W. Muturi1, Scholastica Mathenge1 and Gideon M.

Mutua2 1 School of Medicine, Kenyatta University, P. O Box 43844-00100 Nairobi, Kenya. 2 Department of Obstetrics and Gynecology, Mama Lucy Kibaki Hospital, P. O Box 1278-00515 Nairobi, Kenya.

Received 11 May, 2020; Accepted 10 June, 2020

Surgical site infections account for high mortality, morbidity, and elevated costs

of treatment for surgical patients. The study sought to determine the prevalence

and antibiotic susceptibility patterns of bacterial isolates from postoperative

wound infections among patients attending Mama Lucy Kibaki Hospital. A

cross-sectional descriptive study was carried out between October 2018 and

March 2019. It included patients of all age groups with surgical site infections

following general, obstetrics, and gynecological surgeries. Pus swabs were

obtained aseptically from 58 consented patients with clinical evidence of

surgical site infections. Gram stain, culture, biochemical tests, and antibiotic

susceptibility tests were done for each pus swab. The preponderant isolate was

Staphylococcus aureus (28.2%) followed by Escherichia coli (15.4%). Whereas

Methicillin-resistant S. aureus accounted for 65.4% (n=17) of the total

Staphylococcus species. Chloramphenicol was the most sensitive drug to all the

bacteria isolates. Ampicillin and amoxycillin recorded resistance rates >90%

against Gram-positive and Gram-negative bacteria. The majority of the Gram-

negative rods were highly resistant. Hence, this calls for continuous monitoring

of the susceptibility patterns to determine the profile of surgical site infections

bacteria isolates found in the hospitals.

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Appendix VII- Culture plates

Bacteria swarming on blood agar

Lactose fermenter on MacConkey agar

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Appendix VIII- An algorithm for identifying Gram-positive bacteria

Gram-positive

Cocci Rods

Aerobes Anaerobes

Aerobes Anaerobes

Peptostreptococcus

Catalase test Bacillus species Clostridium species

Catalase positive examples Catalase negative examples

Staphylococcus species Streptococcus species

Coagulase test Hemolysis on blood agar Gamma hemolysis

Enterococcus species

Coagulase positive Coagulase negative Beta hemolysis Alpha hemolysis

Examples S. aureus S.epidermidis S.pyogene S.pneumoniae and viridans

S.saprophyticus

Optochin test

Novobiocin test

Optochin sensitive Optochin resistant

S.pneumoniae S.viridans

Novobiocin sensitive Novobiocin resistance

Staphylococcus epidermidis Staphylococcus saprophyticus

75

Appendix IX - Preparation of MacConkey agar

a) Suspend 49.53 grams of the MacConkey powder in 1000 mls of distilled water

b) Heat to boiling to completely dissolve the medium

c) Autoclave the medium at 121oC for 15 minutes

d) Cool the medium to 45-50oC, mix well and then pour it into sterile petri dishes

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Appendix X- Preparation of Blood agar

a) Suspend 40 grams of the powder in 1000mls of distilled water

b) Heat to boiling to completely dissolve the medium

c) Autoclave the medium at 121oC for 15 minutes

d) Cool the medium to 45-50oC and aseptically add 5% sterile defibrinated blood

e) Mix well and pour the medium into sterile petri dishes

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Appendix XI- Preparation of Mueller Hinton

a) Suspend 38 grams of the powder in 1000 mls of distilled water

b) Heat to boiling to completely dissolve the medium

c) Autoclave the medium at 121oC for 15 minutes

d) Cool the medium to 45-50oC, mix well and then pour it into sterile petri dishes

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Appendix XII - Mama Lucy hospital research approval letter