RESEARCH PROPOSAL

BACTERIAL ISOLATES AND ANTIMICROBIAL SUSCEPTIBILITY PROFILES IN

WOUND SWABS FROM POLOKWANE MANKWENG HOSPITAL COMPLEX,
LIMPOPO SOUTH AFRICA

By

Mudau I (201319056), Shithlangu MJ (201306391), Londani P (201320172), Phetla SK

(201322774), Matihagane MP (201324030), Maswanganyi J (201321798)

RESEARCH PROPOSAL

Submitted in partial fulfillment of the requirements for the degree of

BACHELOR OF SCIENCE

in

MEDICAL SCIENCES

in the

Department of Pathology and Medical Sciences

FACULTY OF HEALTH SCIENCES

(School of Health Care Sciences)

at the

UNIVERSITY OF LIMPOPO

Supervisor: Ms NTC Maguga-Phasha

Co-supervisor: Dr ME Makgatho

Mrs F Mashinya

2016

I
TABLE OF CONTENTS

TERMINOLOGY……………….………………………………………………………………
(iii) ABBREVIATIONS…………………………………………………………………………….
(iv)

1. INTRODUCTION……………………………………………………………………….…..1
1.1. Background………………………………………………………………………..… 1
1.2. Problem statement…………………………………………………………………...3
2. LITERATURE REVIEW………………………………………………………………..….4
2.1. Categories of wound infection………………………………………………..........4
2.2. Bacteriome common in wound infections…………………………………….......4
2.3. Antimicrobial agents used in the treatment of wound infections…………...…..4
2.4. Factors predisposing patients to wound infection in hospitals………………....4
3. Purpose of the study……………………………………………………………….............8
3.1. Aim……………………………………………………………………………………..8
3.2. Objectives…………………………………………………………………………… 8
3.3. Conceptual framework…………………………………………………………...….8
4. METHODOLOGY……………………………………………………………………...…..10
4.1. Study design…………………………………………………………………………10
4.2. Study area…………………………………………………………………………….10
4.3. sampling……………………………………………………………………………..10
4.4. Study population……………………………………………………………………10
4.5. Sample size………………………………………………………………………….10
4.6. Data collection…………………………………………………………………….. 10
4.7. Laboratory procedure……………………………………………………………… 10
4.7.1 Culturing……………………………...……………………………………….10
4.7.2 Microbial identification and confirmation (see appendix 2)………………10
4.8. Antimicrobial susceptibility testing (see Appendix 3)……………………………10
4.9. Data analysis…………………………………………………………………………10
4.10. Reliability and validity……………………………………………………………….10
4.11. Bias……………………………………………………………………………………10
5. Ethical consideration and approval (see appendix 4)………….. ……………………13
5.1.1. Informed consent and voluntary participation………………………….………13
5.1.2. Anonymity and confidentiality………………………………………….………..13
5.1.3. Questionnaire ( see appendix 5)…………………………………….………….13
6. Significance of the proposed research………………………………………………..13

REFERENCES……………………………………………………………………….……...14

APPENDIX………………………………………………………………………………..19
Appendix 1: Gram staining…………………………………………………………….…19

II
Appendix 2: Biochemical tests…………………………………………………....19
Appendix 3: E-test…………………………………………………………………….……..19
Appendix 4: consent form …………………………………………………..……..19
Appendix 5: QUESTIONAIRE…………………………………………………….……….19

III
TERMINOLOGY

Superbugs – are microorganisms that are multi-drug resistant (online Mayo Clinic)

Wound - Wound is a disruption of normal anatomic structure and function of the living
tissue (Leaper et al., 1998).

Wound infection - Infection of wound is the successful invasion, proliferation by one or

more species of microorganisms anywhere within the body’s healthy tissues and
sometimes resulting in pus formation (Mama et al., 2014)

IV
ABBREVIATIONS

API - Analytical Profile Index

ATP - Adenosine triphosphate

E-test - Epsilometer test

ICU - Intensive Care Unit

MBC - Minimum bactericidal concentration

MIC - Minimum Inhibitory concentration

NHLS - National Health Laboratory Service

PCR- Polymerase Chain Reaction

PMHC - Polokwane Mankweng Hospital Complex

SPSS - Statistical Package for Social Science

TREC - Turfloop Research Ethics Committee

WHO- World Health Organization

V
1. INTRODUCTION

1.1. Background

Wound infections have been regarded as the most common nosocomial infection and
present a major challenge to patients, health care staff and the general health care
system in terms of costs and management (Dionigo et al., 2001; Sienkiewicz et al.,
2014). The majority of these infections present in a number of clinical conditions like
non-healing and burn wounds, injuries due to trauma, existing chronic infections like
diabetic foot and pressure ulcers as well as post-operative and surgical wounds (Kagan
and Smith, 2000; Bowler et al., 2001; Benwan et al., 2012; Braga et al., 2013; Aisha et
al., 2013). Wound infection can results in many complications from non-healing wounds,
wound necrosis, spread of infection to the nearest healthy tissues, bacteremia,
septicemia to amputation.

When the various clinical situations are presented, the skin loses its function as a first
line barrier to infections acquired in the hospital or patient’s own endogenous and
exogenous flora (Bhatt and Lakhey, 2007; Nagoba et al., 2010). Infection of wound is
the successful invasion, proliferation by one or more species of microorganisms
anywhere within the body’s healthy tissues and sometimes resulting in pus formation
(Mama et al., 2014).The bacterial agents responsible for wound infections are derived
from aerobic Gram-positive pathogens like Staphylococcus aureus, and Streptococcus
pyogenes as well as aerobic Gram-negative bacteria including Escherichia coli,
Klebsiella pneumonia, Pseudomonas aeruginosa and Acinetobacter baumanii
(Sienkiewicz et al., 2014; Ozumba and Jiburum, 2000; Frykberg, 2003). Anaerobic
Gram negative bacteria such as Bacteroides fragilis as well as anaerobic Gram positive
bacteria like Clostridium difficile and Propionic spp. are also causative agents of wounds
(Mahmoud et al., 2013).

Some of these bacteria are ‘hospital acquired superbugs’, with Acinetobacter baumanni
being the most important superbugs responsible for wound infection in diabetic patients
(Al-Sultan, 2014). According to online Mayo Clinic, superbugs are microorganisms that
are multi-drug resistant. Multi-drug resistant superbugs are increasingly occurring
worldwide because of misuse of different antibiotics, this includes Mycobacterium
tuberculosis, Enterococcus faecium, Enterobacter cloacae, Klebsiella pneumoniae,
Staphylococcus aureus, and Pseudomonas aeruginosa (WHO.,2004, Mahar BB.,2010,
Virk A et al.,2000). Other superbugs are carbapenems resistance bacteria which
includes Proteus app, Citrobacter freundii, Klebsiella oxytoca, (Karthikeyan K K et al.,
2010). Due to hospital acquired superbugs many people lose their lives, parts of their
body and this is becoming a major public health problem (piterson LR et al., 2000).

1
A number of factors expose patients to infections. In addition to the loss of skin due to
trauma; extended hospital stay increases chances of acquiring infections in health care
setups, routine use of invasive procedures and an immunosuppressive effect of trauma
due to burn injury (Ichida et al., 1993; O’Sullivan and O’Connor, 1997; Ashariak et al.,
2004). Moreover, wound infections with multiple micro-organisms (polymicrobial) as well
as an added problem with drug resistance impacts on drug policy formulation by health
care staff (Roberts et al., 2008). The current spread of multi-drug resistant bacterial
pathogens has added a new dimension to the problem of wound infections (Alharbi and
Zayed, 2014). This is particularly worse in resource-poor countries where sales of
antibiotics are poorly controlled (Onule, 1997).

Previously, two studies were conducted in University of Limpopo using wound swabs
from Polokwane Central NHLS. The first study was identifying and speciating
Staphylococus using real time PCR and their antimicrobial susceptibility profiling. The
second study was based on identification of bacterial isolates and antimicrobial
susceptibility profiling in wound swabs from the same facility. From these studies the
observed short comings were lack of data on; site of swab collection, treatment history,
socio-demographic information, type of wound (“clean” and non-healing) and culturing
swabs under aerobic and anaerobic conditions. Moreover, the studies employed an
outdated procedure (Kirby-Bauer disc diffusion assay) and only first generation
antibiotics were used to determine the antibiotic susceptibility profiles of the bacteria.

The proposed study will then identify bacterial species in wounds from patients in
Polokwane Mankweng Hospital Complex, Limpopo Province, South Africa and also
determining their antimicrobial susceptibility profiles/patterns.

2
1.2. Problem statement

Wound infections had been regarded as the most common nosocomial infection and
present a major challenge to patients, health care staff and the general health care
system in terms of costs and management. Some of these wound infections are caused
by the hospital acquired superbugs. There is lack of information about bacterial species
responsible for wound infection and their susceptibility profile here in Limpopo because
the two studies which were previously conducted by the University of Limpopo were not
published because of their short coming. The proposed study will disclose bacterial
species responsible for wound infection, different superbugs and their antimicrobial
susceptibility profiles, using clinical samples collected from PMHC.

2. LITERATURE REVIEW
3
 2.1. Literature review

2.1.1. Categories of wound infection

Wound is a disruption of normal anatomic structure and function of the living tissue
(Leaper et al., 1998). Wounds can be classified as acute or chronic. A wound can either
be surgical, traumatic, burns, bite wounds, cuts, grazes, ulcer and cancer wound
depending on the cause. Patients with burns are usually hospitalized for a long period of
time mainly because of the size of wound (Poh and Yeo, 1993).

2.1.2. Bacteria common in wound infections.

Compared to surgical wounds, burns are more suitable site for bacterial colonization
(Agnihotri et al., 2004). The most common micro-biota that colonizes burns is
Pseudomonas aeruginosa (Nicholas et al, 2013). The microbial aetiology of ulcer wound
infection is usually complex (Shankar, 2005). Different organisms are mostly found in
large numbers in the surgical and trauma wounds. According to Sienkiewics et al.,
2014; Frykberg, 2003; Mahmoud et al., 2013; Bowler et al., 2001; Bowler and Davies,
1999, table 1 represent the most common bacteriome in wound infection.

Table 1: Bacteria common in wound infection

Aerobic isolates Anaerobic isolate

Gram positive Gram positive
Staphylococcus aereus Peptostreptococcus spp.
Coagulase negative staphylococci Clostridium spp.
Streptococcus pyogenes Propionibacterium spp.
Gram negative Actinomyces spp.
Escherichia coli Eubacterium spp.
Klebsiella pneumonia Gram negative
Citrobacter spp. Bacteriodes fragilis
Enterobacter spp. Prevotella spp.
Pseudomonas spp. Veilonella spp.
Serratia marcescens Porphyromonas
Morganella. Morganii Fusobacterium
Acinetobacter spp.
Candida spp.

2.1.3. Antimicrobial agents used in the treatment of wound infections

4
 Classes of Examples of General mechanisms of action of
antimicrobial antimicrobial agents antimicrobial agents.
agents
Beta-lactams Vancomycin Inhibition of Cell Wall Synthesis.
Carbapenemes Meropenem Inhibition of cell wall synthesis
Aminoglycosides Amikacin Inhibition of protein synthesis
Fluroquinolone Ciprofloxacin Inhibition of nucleic acid replication
and transcription
Glycylcycline Tigecycline Inhibition of protein synthesis
Polymyxin Colistin Inflicting injury to the plasma
membrane
Folate pathway Trimethoprim- Interfering with folic acid
inhibitor sulfamethoxazole metabolisim

2.1.4. Factors predisposing patients to wound infection in hospitals.

2.1.4.1. Extended hospitalization.

Extended hospital stay exposes wounded hospitalized patients to wound infections,

during change of bandages (Ichida et al., 1993; O’Sullivan and O’Connor, 1997;
Askariak et al., 2004). During wound treatment and bandage exchange wounds are
expose to air and bacteria are also found free living in the air, which provide them with
the opportunity to colonize wounds.

2.1.4.2. Skin loss and wound size.

Skin loss and the size of the wound is directly proportional to wound infection
opportunity (according to Ichida et al., 1993; O’Sullivan and O’Connor, 1997; Askariak
at al., 2004). The greater the size of the wound and the skin lost during the incidence
that resulted in wound the greater the chance of wound infection. The smaller the size
of the wound and skin loss the lesser the chance of infection.

2.1.4.3. Anaemia and ischemia.

According to Mathew et al., 2015 anaemia followed by hypoalbuminaemia were the two
important risk factors for post-operative wound infection in emergency surgeries.
Reduced blood supply to the site of the wound reduces the supply of molecules
responsible for wound healing and immunity against colonisation by microbes, making
wounds more susceptible to infections.

2.1.4.4. Wound cleaning.

5
 Use of bathwater only without disinfectants exposes wounds to infection with aerobic
gram-negative rods such as pseudomonas (Sienkiewicz et al., 2014; Ozumba and
Jiburum, 2000; Frykberg, 2003; Kirby Mazuski, 2009), while use of disinfectants to clean
the wounds reduces the chance of wound infections.

2.1.4.5. Age.

Age is inversely proportional to wound healing, but directly proportion to wound

infection opportunity (Guo and DiPietro, 2010). In children, due to high production of
growth factors, wounds heal at a faster rate and reduces the chance of infection (Guo
and Dipietro, 2010; Emery et al., 2005; Keylock et al., 2008; Hardman and Ashcroft,
2008). In elders, due to low productions of growth factors, wound healing is temporarily
delayed increasing the chance for wound infection (Gosain and DiPietro, 2004; Keylock
et al., 2008; Swift et al., 2001).

2.1.4.6. Stress.

Stress causes a substantial delay in wound healing and deregulation of the immune
system that can result in suppressed immunity, making wounds a suitable site for
infection (Guo and Dipietro, 2010; Kiecolt-Glaser et al., 1995; Marucha et al., 1998;
Sternberg, 2006; Godbout and Glaser, 2006; Boyapati and Wang, 2007).

2.1.4.7. Oxygenation.

Oxygenation prevents wound infection by inducing re-epithelialisation, enhancing

collagen synthesis and promoting wound contraction (Bishop, 2008; Rodriguez et al.,
2008). While deoxygenation of wound impair healing resulting in hypoxic chronic
wounds, which are suitable site for anaerobes colonization (Bishop, 2008; Rodriguez et
al., 2008; Guo Dipietro, 2010).

2.1.4.8. Alcohol consumption and smoking.

Alcohol consumption and smoking impairs wound healing and increase incidence of
infections (Gentilello et al., 1993; Szabo and Mandrekar, 2009; Siana et al., 1989;
Jensen et al., 1991; Ahn et al., 2008; Guo and Dipietro 2010). Other than delayed
wound healing, patients who smokes shows an increase in a variety of complications
such as wound rupture, anastomotic leakage, wound and flap necrosis, epidermolysis,
and a decrease in the tensile strength of wounds which makes wounds more
susceptible to infections(Chan et al., 2006; Ahn et al., 2008). Alcohol consumption
reduces host’s resistance to infection, and alcohol intoxication during injury increases
susceptibility to infection in the wound (Choudhry and Chaudry, 2006; Guo and Dipietro,
2010).

2.1.4.9. State of immunity.

6
The immune system of immune-competent prevents wound infection by killing all the
microbes that contaminate the wound. Wound infection is more common ion immuno-
compromised patients (Guo and Dipietro, 2010).

2.1.4.10. Nutritional state.

Deficiency of nutrients such as ATP, carbohydrate, protein, fat, vitamin and minerals
essential for cell division and differentiation, impairs wound healing and can suppress
the immune system of a patient (Arnold and Barbul, 2006). Malnutrition patients are
more susceptible to wound infections.

2.1.4.11. Diabetes.

Diabetic patients have impairments in healing of acute wounds, and are prone to
develop chronic non-healing diabetic foot ulcers (Guo and Dipietro, 2010). Development
of chronic non-healing diabetic foot ulcers is associated with a decrease in
neuropeptides, hypoxia, dysfunction in fibroblasts and epidermal cells, impaired
angiogenesis and neovascularization and decreased host immune resistance
(Galkowska et al., 2006; Sibbald and Woo, 2008; Guo and Dipietro, 2010). Decrease in
host immune resistance makes diabetic patients more susceptible to wound infection.

2.1.4.12. Obesity.

Obese individuals frequently face wound complications, including skin wound infection,
dehiscence, hematoma and seroma formation, pressure ulcers, and venous ulcers
(Wilson and Clark, 2004; www.cdc.gov/nccdphp/dnpa/obesity). If a wound is located at
skin folds, chance of wound infections are high since skin folds harbours micro-
organisms (Guo and Dipietro, 2010).

3. Purpose of the study

7
3.1. Aim.

The aim of the study is to identify bacterial isolates and to determine their antimicrobial
susceptibility profiles in wound swabs from the Polokwane Mankweng Hospital
Complex, and also to identify factors associated with wound infection.

3.2. Objectives.

a. Identify bacterial species in wound swabs cultured on their respective agar plates by
Gram staining and biochemical tests.

b. Determine the antimicrobial sensitivity profiles of the bacterial species using the E-
test (MICs and MBCs).

Research question

 Which bacterial species are commonly found in wounds and their antimicrobial
susceptibility profiles?

 What is the relationship between wound infection and factors that predispose
patients to wound infection?

1.3.4. CONCEPTUAL FRAMEWORK

8
socio-demography, treatment
history Culture in lab media: culture Microscopic Identification of
and in aerobic and anaerobic the bacteria cultured from
Collections of wound swabs conditions wound swabs
from PMHC

Confirmation of bacterial Antimicrobial susceptibility

identity by biochemical tests testing

9
4. METHODOLOGY

4.1. Study design.

Descriptive cross-sectional study will be conducted from Polokwane Mankweng Hospital

Complex. Cross-sectional study involves collection of samples once from the patients.

4.2.Study area

Polokwane Mankweng hospital complex

4.3. Sampling

Simple random sampling technique will be used

4.4 Study population

Patients with wounds admitted in Polokwane hospital Mankweng Complex, Limpopo

South Africa.

4.5 Sample size

4.6 Data collection

Questionnaire will be used to collect socio-demographic data as well as treatment

history. Clinical samples (swab, gauss and aspirates) will be collected from various
wards (Trauma, Surgical, ICU, Internal Medicine and Maternity wards) by qualified
hospital personell/staff.

4.7 Laboratory Procedure

4.7.1 Culturing

The samples will be cultured on blood, chocolate and MacConkey agar plates. The
blood and McConkey agar plates will be incubated aerobically at 37 0C for 24-48 hours,
and chocolate agar plate will be incubated in CO2 at 370C for 24-48 hours.

4.7.2 Microbial identification and confirmation (see Appendix 1 and 2).

The positive culture will be identified by their characteristic appearance on their

respective media. Biochemical tests will be used to confirm Gram stain reaction using
standard procedures. API (analytical prolife index) test will also be performed.

4.8 Antimicrobial susceptibility testing (see Appendix 3).

Antimicrobial susceptibility tests will be performed on Mueller-Hinton agar plate using E

– test method to determine minimum inhibitory concentrations (MICs) and minimum

10
bactericidal concentrations (MBCs) of the antibiotic used according to the
manufacturer’s instructions (Davies Diagnostics, Johannesburg, South Africa).

The antibiotics that will be used for bacterial isolates antimicrobial susceptibility profiling
are 3rd generation antibiotics listed in the following table.

Antibiotics
Beta – lactams Carbapenems:
 Amoxicillin.  Imipenem.
 Vancomycin.  Doripenem.
 Cefoxitin.  Ertapenem.
 Cefuroxime.  Meropenem.
 Piperacillin – tazobactam.
 clavulanic acid.
 Cefotaxime.
Aminoglycosides: Polymyxins.
 Gentamicin.  Colistin.
 Amikacin.  Bacitracin.
 Polymyxin B.

Fluroquinolones. Folate pathway inhibitor

 Ciprofloxacin.  Trimethoprim – sulfamethoxazole.
 Orfloxacin.
 Levofloxacin.
 Moxifloxacin.
 Norfloxacin.

Nitrofuran
 Nitroflurantion.

4.9 Data analysis

Data will be captured using Microsoft excel and analyzed using Statistical Package for
Social Sciences (SPSS). Data will be presented as descriptive statistics indicating
percentages of recorded events. In statistical modeling, regression analysis will be used
for estimating the relationships among variables. It includes many techniques for
modeling and analyzing several variables, when the focus is on the relationship
between a dependent variable and one or more independent variables (or 'predictors').

4.10 Reliability and validity

11
Reliability is the extent to which an experiment produces the same result on repeated
trials. Reliability will be ensured as follows: Mueller-Hinton agar plates (Oxoid,
Basingstoke, and Hampshire, UK) and the E-test will be used according to the
manufacture’s instruction.

Validity is the extent to which any measuring instrument measures what it is intended to
measure (Ranjbar, 2014). Quality control will be performed on all the machines to
ensure accurate results. Instruments that will be used for culturing, incubation and
sterilization will be used according to the manufacturer’s instructions.

4.11 Bias

Bias is a form of systematic error that can affect scientific investigation and distort the
measurement process. (Varadhan et al., 2010). Random sampling technique will used
in order to avoid bias.

12
5. Ethical considerations and Approval

5.1. Approval

The research proposal will be submitted to Turfloop Research Ethics Committee

(TREC) at University of Limpopo for ethical approval, and the Provincial Department of
Health for approval to collect samples in terms of Health act 61 of 2003.

5.2. Informed consent and voluntary participation (see appendix 4).

Informed consent is defined as the participation’s institutionally or illegally effective

authorization for a professional to perform a procedure on him after expressing the
understanding (Carmel and Joffe, 2005). In this study aim objective and procedures of
the study will be explained to health care workers and patients and they will be given
opportunity to ask questions and we will make sure all participants join voluntary.
Consent form will be given to patient to sign.

5.3. Anonymity and confidentiality.

Confidentiality means not discussing information provided by individual with others,

while anonymity means presenting research findings in ways that ensure individual
cannot be identified (Wise et al., 2006). In the present of study anonymity will be
ensured by using research identifiers and the names of participants will be protected to
ensure confidentiality. The clinical laboratory information and health status of the study
participants will not be revealed.

6. Significance of the proposed research.

This study will add knowledge about bacterial species responsible for wound infection
and their antimicrobial susceptibility profiles, such that the problems caused by wound
infections to patients, clinicians and the general health care system in terms of cost and
management can be reduced.

13
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APPENDICES

Appendix 1: Gram staining.

Procedure:

The smear on a glass slide is covered with few drops of one of the primary stains.
Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders
all the bacteria uniformly violet. After a minute of exposure to the staining solution, the
slide is washed in water. The smear is treated with few drop of Gram's Iodine and
allowed to act for a minute. This results in formation of a dye-iodine complex in the
cytoplasm. Gram's iodine serves as a mordant. The slide is again washed in water and
then decolorized in absolute ethyl alcohol or acetone. A mixture of ecetone-ethyl alcohol
(1:1) can also be used for decolorization. The process of decolorization is fairly quick
and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and
when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and
acetone acts more slowly than pure acetone. Decolorization is the most crucial part of
Gram staining and errors can occur here. Prolonged decolorization can lead to over-
decolorized smear and a very short decolorization period may lead to under-decolorized
smear. After the smear is decolorized, it is washed in water without any delay. The
smear is finally treated with few drops of counterstain such as dilute carbol fuchsin,
neutral red or safranin. The slide is washed in water; excess water is removed using a
blotting paper, dried in air and heat fixed before observing under microscope. Those
bacteria that hold on to primary dyeiodine complex and remain violet are called Gram
positive and those which get decolorized and subsequently take up counterstain
(pink/red) are called Gram negative. Basic fuchsin (present in dilute carbol fuchsin)
stains many Gram negative bacteria more intensely than does safranin, making them
easier to see. Some bacteria which are poorly stained by safranin, such as
Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by
basic fuchsin. In order to ascertain if the staining procedure was satisfactorily
conducted, a control smear of known Gram positive organism (e.g., Staphylococcus
aureus) and a known gram negative organism (Escherichia coli) must be stained
simultaneously. While the fibrin in a clinical specimen may appear gram positive, the
pus cells and epithelial cells are always gram negative

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Appendix 2: Biochemical tests

Analytical profile index

Single isolated colony (from a pure culture) will be suspended in distilled water. API20E
Biochemical Test Strip which contains dehydrated bacterial media/bio-chemical
reagents in 20 separate compartments will be used. API20E Biochemical Test Strip is
commercially available. (Bacteria will react with them and will give different colors which
will help to identify bacteria to the species level). Pasteur pipette will be used to fill up
(up to the brim) the compartments with the bacterial suspension. Sterile oil will be added
into the ADH, LDC, ODC, H2S and URE compartments. Drops of water will be poured
in the tray followed by API Test strip and close the tray. The tray will be marked with
identification number (Patient ID or Organism ID), date and initials. The tray will be
incubated at 37oC for 18 to 24 hours.

Appendix 3: E-test
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PROCEDURES:

Inoculum Preparation

The E-test package from the freezer (-200C) will be removed at least 30 minutes before
required. 3 or 4 individual test strain colonies will be emulsified and transferred to a tube
of saline. Turbidity of inoculum will be compared to that in the 0.5 McFarland standards
and adjust it to match that standard.

Inoculation in Muller Hinton Agar

Sterile cotton swab will be dipped into the inoculums and pulled out slightly, and rotated
several times against the inside of the tube above the fluid level to remove excess
liquid. The swab will be streaked over the entire surface of the agar plate by rotating the
plate approximately 600C. Inoculation will be completed by running the swab around the
rim of the agar. The lid of the plate ajar will be left for 5 minutes (no more than 15
minutes) to allow any excess moisture to be absorbed before applying strips.

NOTE: Swab plate within 15 minutes of preparing the adjusted inoculums

Application of E-test strips:

E-test package will be opened by cutting package along the broken line and applied to
agar surface using forceps (or E-test applicator if available). The strip will be placed with
the ‘E end’ at the edge of the plate and with the scale visible (i.e. facing upwards).
Templates will be used to position 4 to 6 strips onto a 150 mm plate or one (seldom two)
strips onto a 90 mm plate. Do not remove or replace a strip once it has touched the
agar. the entire procedure will be repeated also for Quality control Strain (E. coli ATCC
25922). Plates will be incubated at 370 C for 18-24 hrs.

Appendix 4: Ethical consideration

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CONSENT FORM

REASEARCH PROJECT AT UNIVERSITY OF LIMPOPO

TURFLOOP CAMPUS

UNIVERSITY OF LIMPOPO (consent form in English)

Name of project

Bacterial isolates and antimicrobial susceptibility profiles in wound swabs from

Polokwane Mankweng Hospital Complex, Limpopo South Africa

I heard the aim and objectives of the proposed study and was provided the opportunity
to ask questions and given time to rethink the issue. The aim and objectives of the study
are clear to me. I have not been pressurized to participate in any way.

I understand that participation in this project is completely voluntary and that I may
withdraw from it at any time and without supplying reasons. This will have no influence
on the regular treatment that holds for my condition neither will influence the care that I
receive from my regular doctor.

I know that this project has been approved by the Turfloop research and ethics (TREC),
University of Limpopo. I am fully aware that the results of this project will be used for
scientific purpose and may be published. I agree to this, provided my privacy is
guaranteed.

I hereby by give consent to participate in this project.

Name of patient/ volunteer signature of patient

……………………… ………………….. …………………….

Place. Date Witness

…………………………………………………………………………………………………

Statement by the Researcher

I provide verbal and\ or written information regarding this study

22
I agree to answer any future questions concerning the study as the best as I am able.

I will adhere to the approved protocol

………………………………. ……………………… …………… …………………..

Name of researcher signature date place

……………………… ………………. ……….. ……………………

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Appendix 5: QUESTIONAIRE

Wounded patients admitted in Polokwane hospital Mankweng complex, Limpopo

province , south Africa .

Participant’s information

Patient’s identity number:

Age :

Gender :

Ward name :

Wound type :

Wound location :

Duration of wound :

Duration of hospitalization :

Treatment history :

Underlying medical conditions:

24