MZUZU UNIVERSITY

FACULTY OF HEALTH SCIENCES

DEPARTMENT OF BIOMEDICAL SCIENCES

A RESEARCH REPORT

ON

Investigating the effectiveness of Zingiber officinale as antibiotic agent against

Streptococcus pneumoniae and Staphyloccocus aureus

By

Shadreck Kachembwe Phiri

(BSBS/2C/16/15)

([email protected])

SUPERVISOR: MASTER CHISALE

July, 2019.
DECLARATION
I, the undersigned, hereby declare that the work presented in this dissertation is entirely mine
except where otherwise attributed to. This dissertation has not been previously submitted to
any examining board or in fulfilment of any course requirement.

Signature: _________________________ Date: ____________________________________

SHADRECK K PHIRI
Researcher

I confirm that the work reported in this dissertation was carried out by this student under my
supervision:
Signature: _________________________ Date: ____________________________________
MR. M. CHISALE

i
DEDICATION
To my mother, Elube Sikelo, without her I could not see this world.

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ACKNOWLEDGEMENTS
I thank God Almighty for everything; He is the reason I am this far. My sincere gratitude goes
to my supervisor, Mr. MASTER CHISALE, for exceptional mentorship, motivation, and
encouragement, with respect to the writing of this dissertation. I also wish to thank Dr. J.
Kaunda and Dr. J. Affone of Mzuzu University for tireless guidance and synergy in the
production of this dissertation. Thanks also go to the entire Mzuzu University Biomedical
Sciences Department team for the research knowledge and immeasurable expertise rendered to
me during the four years’ period at Mzuzu University. I also acknowledge Luke International
for partly funding this research project during the data collection process. Let me also extend
my gratitude to the following people; Mr Samson Sikelo, Mr L. Kachembwe and Mrs Mhone.
God bless you all.
I acknowledge Research Ethics Committees from Mzuzu Central Hospital, for authorizing this
research to be conducted at their health facility. I also wish to express my gratitude to all
laboratory staff members in microbiology department at MCH for participating in this study. I
am grateful to them for their time in this regard.
Special thanks go to my mother and relatives who assisted in various ways which added value
to this work even by providing mental, emotional, physical, financial and spiritual support.
Your help is greatly appreciated. All classmates and friends at Mzuzu University made my stay
away from home live. I do not take this for granted. God bless you all.

Table of Contents
ACKNOWLEDGEMENTS ................................................................................................................ iii
iii
DEDICATION ............................................................................................................................................ ii
LIST OF ACRONYMS AND ABREVIATIONS .............................................................................. vi
DEFINITION OF TERMS................................................................................................................. vii
LIST OF TABLES ............................................................................................................................... viii
LIST OF FIGURES ............................................................................................................................. viii
ABSTRACT ........................................................................................................................................... ix
1.0 INTRODUCTION........................................................................................................................... 1
1.1 BACKGROUND INFORMATION......................................................................................... 2
1.2 STATEMENT OF THE PROBLEM ............................................................................................ 4
1.3 BROAD OBJECTIVE/AIM ........................................................................................................... 5
1.4 SPECIFIC OBJECTIVES ................................................................................................................. 5
1.5 RESEARCH QUESTIONS ...................................................................................................... 5
1.6 SIGNIFICANCE OF THE STUDY......................................................................................... 5
2.0 LITERATURE REVIEW .............................................................................................................. 7
INTRODUCTION .............................................................................................................................. 7
2.1 EFFECTS OF GINGER ON THE GROWTH OF S. AUREUS.................................................... 7
2.2 EFFECTS OF GINGER ON THE GROWTH OF STREPTOCOCCUS SPP .............................. 8
2.3 THE MINIMUM INHIBITORY CONCENTRATION OF GINGER ....................................... 10
SUMMARY ..................................................................................................................................... 11
3.0 METHODOLOGY ....................................................................................................................... 12
3.1 STUDY DESIGN........................................................................................................................ 12
3.2 STUDY SETTING...................................................................................................................... 12
3.2.1 STUDY AREA .................................................................................................................... 12
3.2.2 SAMPLE SIZE AND SAMPLING METHOD................................................................ 12
3.2.3 INCLUSION AND EXCLUSION CRITERIA ................................................................ 12
3.2.3.1 inclusive criteria .............................................................................................................. 12
3.2.3.2 exclusive criteria.............................................................................................................. 12
3.3 SAMPLE COLLECTION ........................................................................................................... 12
3.4 SAMPLE PROCESSING ........................................................................................................... 12
3.4.1 preparation of ginger extracts .......................................................................................... 12
3.4.2 preparation of concentrations and disc diffusion assays ................................................ 13
3.5 DATA COLLECTION TOOL .................................................................................................... 13
3.6 DATA ANALYSIS ..................................................................................................................... 13
3.7 ETHICAL CONSIDERATION ............................................................................................ 14
3.8 LIMITATIONS OF THE STUDY .............................................................................................. 14
4.0 RESULT PRESENTATION ........................................................................................................ 15

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 4.1 THE CONCENTRATION OF GINGER AQUATIC EXTRACTS AND THE ZONES OF
INHIBITION ON MICROORGANISMS. ....................................................................................... 15
4.2 THE CONCENTRATIONS OF GINGER AQUATIC EXTRACTS ON S. AUREUS ............. 16
4.2 THE CONCENTRATIONS OF GINGER AQUATIC EXTRACTS ON S. PNEUMONIAE ... 17
4.4 THE INHIBITORY CONCENTRATIONS OF GINGER AQUATIC EXTRACTS ON S.
PNEUMONIAE AND S. AUREUS ................................................................................................. 18
CHAPTER 5: ........................................................................................................................................ 19
5.1 DISCUSSION ............................................................................................................................. 19
5.2 CONCLUSION ........................................................................................................................... 21
5.3 RECOMMENDATIONS ............................................................................................................ 22
5.4 AREAS OF FURTHER STUDY ............................................................................................. 22
REFERENCES. ................................................................................................................................... 22
APPENDIX A: PREPARATION OF MEDIA ............................................................................................ 28
Blood agar (BA)........................................................................................................................... 28
Chocolate blood agar ................................................................................................................... 28
Mueller Hinton agar ................................................................................................................... 28
APPENDIX B: DATA COLLECTION TOOL............................................................................................ 28
MICROBIOLOGY LABORATORY REQUEST FORM ...................................................... 29
APPENDIX C: LETTERS ....................................................................................................................... 29
REQUEST LETTER FOR PERMISSION ............................................................................................. 30
APPENDIX D: METHOD FOR SUSCEPTIBILITY TESTING ..................................................... 30

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LIST OF ACRONYMS AND ABREVIATIONS

AMR Antimicrobial Resistance

CDC Centre for Disease Control

DG Dry ginger

ECDC European Centre for Disease prevention and Control

EMEA European Medicine Agency

KCH Kamuzu Central Hospital

MRSA Methicillin Resistance Staphylococcus Aureus

MCH Mzuzu Central Hospital

MIC Minimum Inhibitory Concentration

URT Upper Respiratory Tract

USA United States of America

VRSA Vancomycin Resistance Staphylococcus Aureus

WHO World Health Organisation

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DEFINITION OF TERMS

Chemotherapy Treatment of disease with chemical substance (Scanlon &

Sanders, 2007)

Disk diffusion method An agar diffusion test to determine microbial susceptibility

to chemotherapeutic agents (Hogg, 2005)

Pharmaceuticals Relating to medicinal drugs, or their preparation, use or sales

(Tortora, Funke, & Case, 2010).

Photoprotection process that helps organisms cope with molecular damage

caused by molecular damage (Tortora et al., 2010).

Susceptibility The lack of resistance to a disease (Scanlon & Sanders,

2007).

Synergistic effect The principle whereby the effectiveness of two drugs used
simultaneously is greater than that of either drug used alone
(Talaro & Chess, 2012)

Synthetic drug A chemotherapeutic agent that is prepared from chemicals in

the laboratory (Tortora et al., 2010).

Zone of inhibition The area of no bacterial growth around an antimicrobial agent

in the disk diffusion method (Tortora et al., 2010).

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LIST OF TABLES
Table 1: The concentration of ginger Aquatic extracts and the zones of inhibition on
Microorganisms………………………………………………………….…. 15

LIST OF FIGURES
Figure 1: The concentration of ginger aquatic extracts on S. aureus………………… 16

Figure 2: The concentration of ginger extracts on S. pneumonia………………………..17

Figure 3: The inhibitory concentration of ginger aquatic extracts on S. aureus and S.

Pneumoniae………………………………………………………………18

viii
ABSTRACT
Medicinal plants are plants that contain phytochemical substances which kill harmful
microorganisms. They are used globally in treatment of different infections such as diarrhoea,
dysentery, tuberculosis and other diseases. Studies to screen medicinal plants for their
antimicrobial activity as alternative drug have been done in many countries across the World
and in Africa, but much has not been done in Malawi. Ginger (Zingiber officinale) is one of
the medicinal plants that is used in Malawi as a spice and it has been used to treat diseases such
as arthritis, cramps, rheumatism, sprains, sore throats, muscular aches, pains, constipation,
vomiting, hypertension, indigestion, dementia and fever. The study was conducted to
investigate the effectiveness of ginger as an antibiotic agent against Staphylococcus aureus and
Streptococcus pneumoniae grown in the Northern region of Malawi, Mzuzu. using disk
diffusion method.
Fresh ginger rhizomes were collected, sliced and air-dried for seven days at room temperature,
then pounded into powder. The powder was measured using electronic weighing balance into
80g and soaked in a 100ml distilled water, boiled in a water bath for 30minutes, cooled at room
temperature and filtered through Whatman’s filter paper making a concentration of 80%
(80g/100ml). Different concentrations of 80%, 40%, 20% and 10% were prepared using serial
dilution method. The disks were made using Whatman’s filter paper No 1 and prepared using
a sterile puncher. The disks were soaked into the solutions according to concentration for 24
hours, and the lawn of bacterial isolates were prepared on MHA for S. aureus and Chocolate
agar for S. Pneumoniae. The results of the study showed the potent antimicrobial activity of
the ginger extract against staphylococcus aureus on 80% extracts with the Zone of inhibition
of 12mm and 40% (9mm). there were no antimicrobial activity on streptococcus Pneumoniae.

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1.0 INTRODUCTION
The development of bacterial resistance to current chemotherapeutic drugs has necessitated the
immediate need for search and discovery of alternative drugs particularly from the plant origin
which are readily available and has considerable less side effects (Singh, Tripathi, Srivastava,
Ali, & Rekhi, 2016). There are a huge variety of plants, termed as natural products which are
rich in secondary metabolites and have essential oils for therapeutic importance (Dipankar,
Murugan, & Devi, 2011). Most of these natural products are widely used in everyday life either
directly or indirectly for the treatment of different infectious diseases worldwide.

Natural products are the most important source of drugs against many pathological infections
(Abdalla & Abdallah, 2018). Most natural products with medicinal properties are spices, they
have a unique aroma and flavours which are derived from compounds called phytochemicals
(Joe, Jayachitra, & Vijayapriya, 2009). Phytochemicals are antimicrobial substances present in
the spices which are capable of attracting benefits and repel harmful organisms (Liu, 2004),
they also serve as photo-protectants and responds to environmental changes (Joe et al., 2009).
According to World Health Organisation (WHO), 80% of the world’s emerging population rely
on natural products derived from plants for their initial health care and 35% are mostly from
the developed countries and they use herbs directly or indirectly to maintain good health
(Mahomoodally, 2013). In Asia and Africa, 80% of the population uses medicinal plants for
their initial healthcare (WHO, 2008). Due to the emerging and increase of antimicrobial
resistance (AMR) to current chemotherapeutic drugs prescribed in the hospitals, there is an
urgent need for more research to come up with another drug line for replacement.

AMR has become widespread irrespective of countries’ level of income. In Europe, 25,000
people die every year from antibiotic resistance bacteria (ECDC & EMEA, 2009). Recently,
United States (US) registered more than 2 million illness and 23,000 deaths due to antibiotic
resistance (CDC, 2013). In Africa, AMR is widely increased among children, and in the sub-
Saharan Africa including Malawi, more than 30% of patients with bacterial infections have
AMR bacteria (WHO, 2018). This antibiotic resistance in human pathogenic microorganisms
have been developed due to indiscriminate use of commercial antimicrobial drugs commonly
used in the treatment of infectious diseases (Mohamed, 2013). This situation triggered
scientists to search for new antimicrobial substances from various sources as novel
antimicrobial chemotherapeutic agents particularly from plant origin as the production of

1
synthesised drugs is costly and they produce adverse effects compared to plant derived drugs
(Deen, 2017).

Many investigators have demonstrated the antimicrobial activity of some plants and many
chemical compounds of plant origin that have been proven to possess antimicrobial activity.
zingiber officinale (ginger) being one of the medicinal plants, have been used for a long time
as naturopathy due to its potential antimicrobial activity against different microbial pathogens
(Riaz et al., 2015). Accordingly, medicinal plants could be used as an alternative for the
microorganisms that develop resistance to most antibiotics. The aim of this study is to
investigate the effectiveness of Zingiber officinale as an antimicrobial agent against
streptococcus pneumoniae and Staphylococcus. aureus

1.1 BACKGROUND INFORMATION

Traditional herbal medicines are naturally occurring, plant-derived substances with minimal or
no industrial processing that have been used to treat illness within local or regional healing
practices (Singh et al., 2016). Traditional herbal medicines are getting significant attention in
global health debates. In developed countries, the raw materials for manufacturing essential
drugs are mainly extracted from medicinal plants harnessing its natural property of healing
(Motaleb, 2011), and in countries like German and Canada, atleast 70% of their population
have tried herbal medicines (Mahomoodally, 2013). In Asia, the practice of herbal medicine is
well established and documented, as such they have an international recognition especially
from China and India (CTA, 2007).

The extensive use of traditional medicine in Africa, composed mainly of medicinal plants, has
been argued to be linked to cultural and economic reasons. This is why the WHO encourages
African member states to promote and integrate traditional medical practices in their health
system (WHO, 2008). In some parts of Africa, traditional plants like Garlic is used in the
treatment of upper respiratory tract infections such as cough and sore throat; and
gastrointestinal tract infections (Onianwah & Ho, 2018).

In the sub-Saharan Africa, most people rely on traditional medicine and it is often the first or
last line of defence against most diseases such as headache, coughs, diarrhoea, wound healing
and skin diseases (Cheikhyoussef, Shapi, Matengu, & Ashekele, 2011). In Malawi, traditional
medicine is widely used for the treatment of pneumonia, stomach pain (mangifera indica),
2
prevention of miscarriage (Bauhinia petersiana Bolle), cure for post maturity (Ricinus
communis) and in some parts of the country, medicinal plants are used for the treatment of
infertility and infectious diseases (nyirenda & Maliwichi, 2010). Most of these medicinal plants
that are being used are not scientifically proven, they are used culturally and there is a need of
more research to check for the effectiveness of these herbal medicines against pathogenic
microorganisms.

The use of herbal medicine is increasing with the recognition that there is a challenge in the
treatment of some medical conditions (Kigen, Ronoh, Kipkore, & Rotich, 2013). Ginger is one
of the plants that have been widely used all over the world as medicine (Akintobi et al., 2013).
Since antiquity, it has been used to treat arthritis, cramps, rheumatism, sprains, sore throats,
muscular aches, pains, constipation, vomiting, hypertension, indigestion, dementia, fever and
infectious diseases (Ali, Blunden, Tanira, & Nemmar, 2008). Ginger has direct anti-microbial
activity and thus can be used in treatment of bacterial infections (Islam, Rowsni, & Kabir,
2014). Most studies have been conducted to investigate the effectiveness of ginger against
various Microorganisms, but none has been done in Malawi to investigate its effectiveness
against Staphylococcus Aureus and Streptococcus Pneumoniae which are among the AMR
microorganisms in Malawi (Makoka et al., 2012).

Today, the emerging and increasing AMR has been detected in every pathogenic species and
against all classes of antibiotics globally (Gray & Fitch, 2015). In Europe, the AMR is common
and some pathogens like Methicillin Resistance Staph Aureus (MRSA) and Vancomycin
Resistance Staph Aureus (VRSA) have become resistant to multiple classes of antibiotics
(CDC, 2013). The European commission presented the community strategy against AMR and
proposed to take action in surveillance, prevention, international cooperation and research and
development of new drugs (ECDC & EMEA, 2009). Despite all the strategies, patients are
infected and the AMR trend still increases. In USA, a lot of people die due to the AMR and at
least 2 million illnesses are antibiotic resistance related (CDC, 2013). S. pneumoniae and S.
aureus indicates much percentages in drug resistance.

In Sub-Saharan Africa, AMR is an increasing and is a real threat mostly among children
admitted in hospitals (Williams, Isaacs, & Berkley, 2018). In Malawi, most bacterial isolates
from blood cultures are resistant to first line antibiotics like amoxillin, chloramphenicol, and
cotrimoxazole in the southern region (Musicha et al., 2017). In the central region, S.

3
pneumoniae was found to be resistant to contrimoxizole (Makoka et al., 2012). In order to get
lead of the AMR, WHO launched the implementation of the Global action plan on
antimicrobial resistance which aims at sensitizing farmers and the food industry to stop using
antibiotics routinely to promote growth and prevent disease in healthy animals which can
transmit AMR microorganisms to humans, and the launch of global surveillance in reducing
AMR (WHO, 2017). According to the WHO report on Global Antimicrobial surveillance
system (GLASS), many countries took an initiative towards the strategy. In America, the Latin
American Network for Antimicrobial Resistance Surveillance (ReLSVRA) was enstablished,
and was directed towards improving AMR laboratory surveillance in the America through the
strengthening of laboratory capacity for pathogen identification and Antibiotic Sensitivity
Testing (AST) (ReLavra, 2017). In Europe, they developed Proof of Principle (PoP) projects
which are designed to stimulate blood sampling of patients with suspected bloodstream
infection to support treatment decisions of clinicians, as well as to start assessing the AST of
the main pathogens causing community acquired and hospital acquired infections (WHO
EURO, 2017). In African region, Technical assistants are currently planned for countries to
develop national laboratory AMR capacities in Kenya, Tanzania, South Africa, Zambia,
Zimbabwe, Ethiopia, Chad, Mali, Liberia, Mauritius, Algeria and Burkina Faso (WHO, 2018).
Despite the initiative, Malawi has not yet embarked on the strategy for the prevention and
surveillance on AMR, as such there is a need to find new ways to treat AMR.

1.2 STATEMENT OF THE PROBLEM

The emerging and increasing rate of AMR has now become a global problem (Gray & Fitch,
2015). Antimicrobial agents are now losing their effectiveness facilitated by overuse and
misuse in humans and in the agricultural sector (access to medicine foundation, 2018). As such,
new resistance mechanisms are emerging and spreading globally, threatening our ability to
treat common infectious diseases resulting in prolonged illness, disability and death
(Woolhouse, Waugh, Perry, & Nair, 2016). AMR infections currently claim at least 50,000
lives each year across Europe and the US alone, with many hundreds of thousands more dying
in other areas of the world (O’neill, 2014). S. aureus and S. pneumoniae have become resistant
to Malawian first line antibiotics such as Amoxillin, Chloramphenicol, and cotrixazole
(Makoka et al., 2012).

The cost of health care for patients with resistant infections is higher than the cost incurred to
care for patients with non-resistant infections due to longer duration of sickness, additional
tests performed and the use of more expensive drugs. Many people in developing countries like

4
Malawi have difficulties in accessing medical care due distance and financial problems to buy
synthesized drugs that are on the market which results in high morbidity rate.

WHO proposed strategies against AMR in communities (WHO, 2017). It proposed actions in
the areas of surveillance, prevention, international cooperation & research and development of
new drugs. Despite other countries taking an initiative to curb AMR, Malawi, has not done
much on screening of Medicinal plants for their antimicrobial properties as a step to the
development of new drugs. Due to this, there is a need to investigate the effectiveness of
Ginger, one of the plants commonly found in Malawi

1.3 BROAD OBJECTIVE/AIM

The main objective of this study is to investigate the effectiveness of ginger as an antibiotic
agent against S. pneumoniae and S. aureus.

1.4 SPECIFIC OBJECTIVES

1. To determine the effect of ginger on the growth of S. aureus
2. To determine the effect of ginger on the growth of S. pneumoniae.
3. To determine the Minimum Inhibitory concentration of ginger on S. aureus and S.
pneumoniae.

1.5 RESEARCH QUESTIONS

1. Does ginger have an effect on the growth of S. aureus?
2. Does ginger have an effect on the growth of S. pneumoniae?
3. What is the Minimum inhibitory concentration of ginger on S. Aureus and S.
Pneumoniae?

1.6 SIGNIFICANCE OF THE STUDY

The results of this study may help to reduce the infections caused by AMR in the community.
The discovery has the potential to review new and locally available antibiotic agent against
pathogens that cause serious health problems, as such reduces the number of death. The study
may also help people in the community with low financial status who are affected by AMR
and has prolonged illness to access local antibiotic agents hence reduces the cost of buying
expensive synthesised drugs.

5
The study may help the Ministry of health in the discovery and screening of drugs as
proposed by WHO. The strategies proposed by WHO remain a problem in Malawi and if
Ginger may be found to be effective on S. aureus and S. pneumoniae which are some of the
major AMR microorganisms in Malawi, it can be a start-up of the strategy.

6
2.0 LITERATURE REVIEW

INTRODUCTION
Studies were done in different countries on various ginger extracts and different
microorganisms. The studies reviewed in this chapter are from America, Europe and Africa. In
Malawi, little is known about the effectiveness of ginger and there are no published studies yet
on the investigation of ginger as antibiotic agent against the selected pathogens. The
effectiveness is being observed to vary according to region, bacterial strains, type of ginger
used, extracts and method of preparation of extracts.

2.1 EFFECTS OF GINGER ON THE GROWTH OF S. AUREUS

A prospective study based on laboratory investigation was done in Stamford university,
Bangladesh to determine the antimicrobial activity of soybean oil extracts of dried ginger
powder using agar diffusion assay against different foodborne pathogens namely E. coli, S.
aureus, pseudomonas aruginosa, Vibrio cholerae, Klebsiella spp and salmonella spp. Ginger
mixed oil was boiled in a water bath for 30 minutes, cooled at room temperature and filtered
through whatman’s filter paper and collected in a sterile container. The results showed
variations in the antimicrobial activity of the soybean oil extracts according to the type of
bacterial isolate used. The extracts were found to be sensitive to salmonella spp and klebsiella
spp, but it was less sensitive to S. aureus (Islam, Rowsni, & Kabir, 2014). According to the
study done by Laodheerasiri & Pathirage, (2017), it was found that soybean has antimicrobial
activity against E. coli and S. aureus. So the study was not clear on the antimicrobial activity
of the soybean oil extracts of ginger as the effectiveness was a synergistic effect of soybean oil
and ginger, and the effect of ginger only on selected microorganisms was not determined. In
this regard, the researcher focused on ginger aqueous extracts to investigate the constituents of
ginger antimicrobial properties against S. aureus and S. pneumoniae. The study by
Laodheerasiri & Pathirage, (2017) also found that soybean extracts of ginger at boiling
temperature has potential antimicrobial activity against all selected microorganisms which is
contrary to the study done by Pankaj et al, (2012) which determined the effect of temperature
on antibiotic properties of ginger, and found that boiled ginger lost its effectiveness on
Klebsiella Pneumoniae, E. coli and S. aureus. So the researcher did not boil ginger to the
boiling point of water to avoid denaturing some essential phytochemicals in it.

Another study was done in Nigeria by Akintobi et al., (2013) to investigate the antimicrobial
activity of ginger extracts against Pseudomonas aeruginosa, S. aureus, Proteus mirabilis, E.
coli, Bacillus subtilis and Salmonella typhi using the agar well diffusion method. Aqueous and
ethanol extracts of ginger were used and the results showed variations among isolates in their
7
sensitivity according to extracts. Aqueous extracts of ginger were sensitive to salmonella typhi,
proteus mobilis and S. aureus and ineffective against E. coli, Bacillus subtilis and pseudomonas
aeruginosa. Ethanol extracts were sensitive on proteus mobilis, salmonella typhi and S. aureus.
S. aureus was found to be sensitive to all extracts. As observed in the results, it showed the
different behaviour of isolates in their sensitivity to the different extracts added to their growth
medium. All microorganisms that were not effective on aqueous extracts were also not
effective on ethanol extracts except Pseudomonas aeruginosa which was ineffective in aqueous
extracts only and those which were effective on ethanol extracts were different from those
which were effective on aqueous extracts except S. aureus. The difference between the
ineffectiveness of Pseudomonas auruginosa as compared to the trend of other microorganisms
on ethanol extracts only was not clearly explained and could be attributed to the genetic
variations of the microorganisms and ginger variety. The effectiveness of aqueous extracts
differs with the similar study done by Onianwah & Ho, (2018) which found that the aqueous
extracts were not effective to S. aureus. So this study determined the effect of ginger aqueous
extracts grown in Malawi on the growth of S. aureus from different clinical isolates.

According to the study done by Gull et al., (2012) which was aimed at investigating the
inhibitory effect of Garlic (Allium sativum) and ginger extracts on clinically important drug
resistant pathogenic bacteria, three types of extracts namely; aqueous, ethanol and methanol
extract from each garlic and ginger were prepared separately. The fresh cloves of both ginger
and garlic were washed, peeled, sliced and sundried for seven days. After drying, slices were
ground to fine powder separately using electric blender. It was found that all tested bacterial
strains including S. aureus strains were susceptible to garlic and showed poor susceptibility to
the ginger aqueous solution. This study differs with the results of Akintobi et al., (2013) which
reported that S. aureus was susceptible to aqueous extracts. On sample processing, the ginger
was sundried, and the sun was found to degrade some active chemicals in ginger hence affects
its activity (Sasidharan & Menon, 2010). The poor susceptibility may be due to the degradation
of some chemical compounds, so the researcher in this study used air dried ginger to determine
its effect on the growth of S. aureus.

2.2 EFFECTS OF GINGER ON THE GROWTH OF STREPTOCOCCUS SPP

A study was done in Iraq to evaluate the antibacterial activity of ginger against gram positive
bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumonia,
Streptococcus feacalis, Streptococcus mutanus, Streptococcus feacalis) and gram negative
bacteria (Escherichia coli,Salmonella typhi, Moraxallia catarralis, Pseudomonas aeroginosa,
Proteus mirabilis, Klebsiella pneumonia, Enterobacter spp. Acinetobacter, Serratia spp) using
8
well agar diffusion test. Four ginger products were used to determine the antibacterial activity
of aquatic extracts of fresh ginger, aquatic extracts of powder ginger, crude oil of ginger and
apple vinegar extracts of fresh ginger. Fresh ginger was peeled, sundried, and cut into small
pieces, then grounded using an electric blender and placed in a clean container. preparation of
apple vinegar, ginger extracts was dissolved in 50gram of ginger and sterilised by filtration.
The results showed that the apple vinegar extract of fresh ginger produced the highest inhibition
activity against all tested Microorganisms, the aquatic extracts of ginger showed slight effect
on all microorganisms and crude oil extracts indicated poor sensitivity (Hindi, Al-mahdi, &
Chabuck, 2014). The sensitivity pattern among organisms, streptococcus pneumoniae and
streptococcus epidermidis were more sensitive (35mm) to apple vinegar extracts, and in crude
oil, streptococcus pneumoniae did not indicate any zone of inhibition (0mm) while
Streptococcus epidermidis indicated a slight zone of inhibition (6mm). The difference in S.
pneumoniae and S. epidermidis in crude oil were not reported both being streptococcus spp.
There is a need of more studies on the difference to clear the discrepancy. Gunathilake &
Rupasinghe, (2015) reported that the activity of ginger is highly affected by PH. In the study,
vinegar (acidic) was used and it was found to be more effective. This study used aqueous
extracts to determine the effect of ginger on the growth of S. pneumoniae.

According to the study done by Nader, Ghanima, Ali, & Azhar, (2010) which was aimed at
investigating the antimicrobial activity of ginger extracts (cold water, hot water, ethanolic and
essential oil) against Streptococcus spp., Escherichia coli, Salmonella spp., Klebsiella spp.,
serratia marcescens, Vibrio cholera and S. aureus was investigated using Disc diffusion
method. It was found that cold water extracts were more effective on all tested microorganisms
except Serratia marcescens and Vibrio cholera, hot water extracts were ineffective on all tested
microorganisms, ethanoic and essential oil extracts were effective on all tested microorganisms
except serratia marcescens and Vibrio cholera and the highest zone of inhibition was observed
in streptococcus sp. (28mm and 20mm respectively). The results differ with the study by Malu,
Obochi, Tawo, & Nyong, (2009) which reported that streptococcus sp. was not effective on
cold water extracts. The reason behind the difference might be the difference in the variety of
ginger grown in the two regions. The variations in antibacterial results of ginger could be
attributed to the fact that ginger rhizomes are greatly affected by geographic variations,
environmental conditions, and physiologic factors which influence its bioactive phytochemical
compounds which could alter the quality and quantity of bioactive phytochemical compounds
(Abdalla & Abdallah, 2018). This study was done in Malawi to determine the effectiveness of
ginger grown on selected bacterial strains found at Mzuzu Central Hospital.
9
2.3 THE MINIMUM INHIBITORY CONCENTRATION OF GINGER
A lot of studies have investigated the minimum inhibitory concentration (MIC) of ginger on
various microorganisms. It is important to find the MIC so that the required amount of ginger
is taken for its effectiveness. According to literatures, the concentration varies according to the
extract, type of microorganisms used, bacterial strains and the nature of the study. A study was
done by Rajsekhar, Chandaker, & Upmanyu, (2012) in india. The aim of the study was to
review ginger, garlic and clove (Syzygium aromaticum) as antimicrobial agents. The study was
evaluated against some foodborne bacteria and fungus namely Bacillus cereus, Bacillus
subtilis, Bacillus sp., Staphylococcus aureus, Staphylococcus epidermidis, Listeria
monocytogenes Micrococcus luteus, Escherichia coli, Klebsiella sp. and Pseudomonas
aeruginosa. The antimicrobial activity of cinnamon extract and oil were determined by the agar
well diffusion method. The MIC of ginger on staphylococcus aureus was 20g/100ml (with the
diameter of 10mm) and the zone of inhibition increased significantly as the concentration
increased to 100g/100ml which recorded the highest zone of inhibition of 30mm-32mm.
Streptococcus pyogenes had the lowest zone of inhibition of 19mm at 20g/100ml of water
extracts. Ethanol extracts was found to have the MIC of 20g/100ml (with the zone of inhibition
of 20 and 21mm for staphylococcus aureus and streptococcus pyogenes respectively) and the
highest concentration of 100g/100ml had 30mm zone of inhibition. Various MIC are found on
different extracts for the microorganisms and the minimum concentration depends on the
resistance pattern of the microorganisms in that area. The microorganisms used were isolated
in food which did not go under exposure of antibiotics to mutate, hence cannot be used to
determine the MIC of other organisms. So clinical isolates for different strains were used to be
investigated in order to have proper MIC.

In another study done by Azizi et al., (2015) which was aimed at assessing the effect of different
concentrations of ginger extract on proliferation of Streptococcus mutans and Streptococcus
sanguinis in vitro, serial dilutions of the extracts were prepared in two sets of 10 test tubes for
each bacterium. Different concentrations were set to measure the zones of inhibition and it was
found that the MIC was 0.02mg/mL for S. mutans and 0.03mg/mL for S. sanguins. As observed
in the literature, both microorganisms are in the group of streptococcus sp. and have different
MIC. So there is a need to find out the MIC for other microorganisms in the Streptococcus spp.
to determine their MIC, and this study looked unto the MIC for Streptococcus pneumoniae and
Staphylococcus aureus. In the study, the microorganisms used were control microorganisms
(ATCC 35608 for S. mutans and ATCC 10556 for S. sanguinis) which are standard strains that
does not face the mutation for resistance. So it is difficult to come up with the exact MIC for

10
other strains which undergo mutation. There is a need to come up with different isolates from
different patients to see the pattern of resistance and come up with the MIC.

SUMMARY
The investigation of an effectiveness of Ginger as an antibiotic agent has been studied in
different countries against different microorganisms. From these studies, it indicates that the
effectiveness of this plant product is greatly affected by many reasons such as the method of
extraction, antibacterial assay conditions, genetic variations among bacterial strains and its
sources. The origin of plant sample is also an important factor since plants are affected by
geographic variations, environmental conditions and physiologic factors which influence its
bioactive phytochemical compounds.

11
3.0 METHODOLOGY
This section focused on study design, study setting, sample collection, sample processing,
inclusion and exclusion criteria, data collection, data analysis, quality assurance, ethical
consideration, limitation of the study and dissemination of results.

3.1 STUDY DESIGN

An experimental study design and quantitative research approach was conducted to investigate
the effectiveness of Zingiber officinale as an antibiotic agent against staphylococcus aureus
and Streptococcus pneumoniae.

3.2 STUDY SETTING

3.2.1 STUDY AREA
The study was conducted at Mzuzu Central Hospital (MCH) Microbiology Lab, which is one of
the major referral hospitals in the northern region. Its laboratory receives samples from different
patients in the districts of northern Malawi. MCH Microbiology lab has the capacity to conduct
culture and sensitivity using more sensitive techniques.

3.2.2 SAMPLE SIZE AND SAMPLING METHOD

A convenience (non-probability) sampling technique was adopted to identify number of
isolates to be used (Bluman, 2009). From the samples which were found on stock, 34 samples
were for S. aureus and 12 were for S. pneumoniae. All samples were recruited in the study.

3.2.3 INCLUSION AND EXCLUSION CRITERIA

3.2.3.1 inclusive criteria
All samples identified as S. aureus and S. pneumoniae were included in the study

3.2.3.2 exclusive criteria

All samples identified not to be S. aureus and S. pneumoniae were not included in the study.

3.3 SAMPLE COLLECTION

The fresh forms of ginger were bought from Mzuzu local market, then sealed in a plastic bag for
easy transportation to Mzuzu University, Biology Lab for processing.

3.4 SAMPLE PROCESSING

3.4.1 preparation of ginger extracts
Fresh Ginger rhizomes were sliced, air dried for 7 days at room temperature to avoid degradation
of some chemicals, then pounded into powder and sieved using pestles. The powder was measured
using electronic weighing balance into 80g, and the 80g weighed powder was soaked in 100ml
12
distilled water and boiled in a water bath for 30 minutes, cooled at room temperature and filtered
through Whatman’s filter paper to obtain 80% (80g/100ml) concentration (w/v) (Bishop, Fody, &
Schoeff, 2010). The extracts were collected in a sterile conical flask and stored at 40C for 72 hrs
for further use.

3.4.2 preparation of concentrations and disc diffusion assays

The investigation on different concentrations was done using serial dilution method (Turgeon,
2016). Four test tubes were set labelled 80%, 40%, 20% and 10% (Ebrahimi, Bazargani,
Pourshahidi, Rafiee, & Gavahi, 2012). In each test tube, 1 ml of distilled water was added except
the first one labelled 80%. To the test tube labelled 80%, 1 ml of extracts was added. To the test
tube labelled 40%, 1 ml of the extracts was added as well. Then 1ml of 40% was added to the 20%
test tube, and finally, 1ml of 20% was added to the last 10% test tube to make 10% concentration.

Whatman’s Filter paper No 1 discs (6mm diameter) were prepared using a punch machine and
labelled G1, G2, G3 and G4 for 80%, 40%, 20% and 10% respectively. The discs were wrapped
with aluminium foil in a glass petri dish and autoclave at 1210C for 15 minutes. After sterilization,
the discs were soaked into the solutions according to concentration for 24 hours.

A lawn of each bacterial isolate were prepared on MHA plates for S. aureus and Chocolate Agar
for S. pneumoniae using a sterile cotton swab from the inoculum showing growth of 0.5 McFarland
standard (Appendix D). The discs with different concentrations, using a sterile syringe, were put
on the agar streaked with an organism of interest. The plates were incubated at an average
temperature of 37.0oC for 24 hours. After 24 hours, the plates were measured for the occurrence
of zone of inhibition (Cheesbrough, 2006). Amoxillin (AMC) and Gentamycin (GM) were used
as control antibiotic agents for quality assurance.

3.5 DATA COLLECTION TOOL

Data were collected by measuring the zones of inhibition for each isolate in a media for
different concentrations using a checklist (Appendix B)

3.6 DATA ANALYSIS

The diameter of the zone of inhibition from the media which was measured using a ruler, was
recorded manually in a checklist. Thereafter, this numerical data was compiled and analysed
using a computer package Microsoft Excel 2016 to find mean and frequency which helped in
formulating frequency tables and charts, that show the sensitivity of ginger on selected
microorganisms and the Minimum zone of inhibition which represents the Minimum Inhibitory
Concentration (minimum concentration of prepared extracts) of ginger.

13
 3.7 ETHICAL CONSIDERATION
• Permission
➢ Ethical approval of the research was obtained from Mzuzu University Research
Review committee and from the Mzuzu Central Hospital research committee as well
as National Research Council of Malawi through Mzuzu University.
• Privacy and confidentiality
➢ Names of patients where microorganisms were isolated were not used, however each
sample had an Identification number (ID).
• Quality assurance
➢ Quality was ensured by following and using aseptic techniques to ensure sterility
throughout the study according to approved standard operating procedures at MCH,
microbiology lab.
➢ Each test had a control check for validity of all materials used. Further instructions by the
supervisor were considered and followed accordingly.
• Justice
➢ Rules and regulations for microbiology department, MCH lab were followed and all
materials used during the study were disposed following recommendations provided by the
manufacturer to avoid putting people and the community at risk.

3.8 LIMITATIONS OF THE STUDY

Due to limited resources and lack of funds, the research focused on ginger aqueous extract only
and other extracts like ethanol and methanol were not used.

14
4.0 RESULT PRESENTATION

4.1 THE CONCENTRATION OF GINGER AQUATIC EXTRACTS AND THE ZONES OF

INHIBITION ON MICROORGANISMS.
concentration/antibiotic test microorganisms

S. aureus (mean Z.I) S. pneumonia (mean Z.I)

80% 12 6

40% 9 6

20% 6 6

10% 6 6

Table 1 shows the concentrations of ginger extracts and the diameter of the zone of inhibition
on isolated microorganisms. Z.I represents the zone of inhibition and 6mm is the size of the disk
and mean there were no zone of inhibition.

15
4.2 THE CONCENTRATIONS OF GINGER AQUATIC EXTRACTS ON S. AUREUS
14

12
12
THE HIGHEST ZONE
OF INHIBITION

10
9
ZONE OF INHIBITION (mm)

6 6
6

0
80% 40% 20% 10%
CONCENTRATION (g/100ml)

Figure 1 shows the zone of Inhibition (mm) for different concentrations of ginger aqueous
extracts for staphylococcus aureus. 6mm represents the size of the disk and means there was no
zone of inhibition.

16
4.2 THE CONCENTRATIONS OF GINGER AQUATIC EXTRACTS ON S.
PNEUMONIAE

6 6 6 6
6
NO ZONE OF
INHIBITION

5
ZONE OF ONHIBITION (mm)

0
80% 40% 20% 10%
CONCENTRATION (g/100ml)

Figure 2 shows the zone of Inhibition (mm) for different concentrations of ginger aqueous
extracts for streptococcus pneumoniae. 6mm represents the size of the disk and means there was
no zone of inhibition.

17
4.4 THE INHIBITORY CONCENTRATIONS OF GINGER AQUATIC EXTRACTS ON S.
PNEUMONIAE AND S. AUREUS

14

S. aureus
12
S. Pneomoniae

10
MIC
ZONE OF INHIBITION (mm)

0
80% 40% 20% 10%
CONCENTRATION (g/100ml)

Figure 3 shows the MIC of Staphylococcus aureus and streptococcus pneumonia. 6 represents
the size of the disc and means there is no inhibition.

18
CHAPTER 5:

5.1 DISCUSSION
The antimicrobial activity of ginger is due to phytochemicals and essential oils. The main
factors that determine the antimicrobial activity are the type and composition of the spice,
amount used, type and resistance of the microorganism, pH value and temperature of the
environment (Gull et al., 2012).

5.1.1 EFFECTS OF GINGER ON S. AUREUS

The antibacterial effects of aqueous extracts exhibited appreciable activity on S. aureus. This
could support the use of the plant in the treatment of staphylococcal infections. This is in line
to the findings of Akintobi et al., (2013) who used 20g of ground ginger and dispensed it in
80ml of distilled water and found that S. aureus was sensitive to the ginger aqueous extracts.
Though there is a similarity in the activity of ginger against S. aureus, the study did not explain
the minimum amount of ginger to be taken for its effectiveness against S. aureus, so it is
difficult to conclude its effectiveness without considering the amount required for efficiency.
The sensitivity of ginger on S. aureus may be due to the fact that the plant extracts might have
provided antimicrobial components that overcame the permeability barrier provided by the cell
wall or cell membrane of the bacteria as asserted by Wei et al, (2008). According to the results
in the figure 1, taking a considerable amount of ginger will increase its effectiveness as the
effectiveness is more in 80% (g/100ml) and decreases as the concentration decreases to 40%.
At 20%, there is no any zone of inhibition, meaning its reduced intake will not have the activity.

The effectiveness of ginger may be due to phytochemicals such as saponin, flavonoids and
alkaloids that are found in zingiberene, the main constituent of ginger, which were reported to
have antifungal and antibacterial activities in vitro (Rajsekhar, Chandaker, & Upmanyu,
2012b) and so effective in combating specific type of diarrhoea which is leading cause of death
in infants in developing countries. Moreover, it has been found that ginger is effective in
treating nausea caused by sea sickness, morning sickness and chemotherapy (Suzanna & Zick
2011). Though the results of this study discovered that the Zone of inhibition for
Staphylococcus aureus was 12mm at 80% concentration and decreased considerably to 10mm
at 40% concentration, other studies reported that ginger is not effective on S. aureus. Onianwah
& Ho, (2018) heated the ginger water extracts to boiling temperature, and the precipitated white
deposits were re-suspended in 1ml of distilled water. Both the supernatant and the re-suspended
deposits were tested for antibacterial activity, and found that the ginger extracts were not
19
effective on S. aureus. This method could not be used to conclude that ginger did not have the
antibacterial activity since the extracts were heated which might have the possibility of
denaturing some essential phytochemicals which have the activity. As observed in figure 1,
there is a decrease of effectiveness as the concentration decreases, so the difference in results
may be that the amount of ginger used was not enough to inhibit the growth of S. aureus

5.1.2 EFFECTS OF GINGER ON S. PNEUMONIAE

Though ginger has been found to contain some antibacterial phytochemicals useful against S.
aureus, it has been found to have no effect on S. pneumoniae. This may imply that ginger
concentrations using distilled water as a solvent and diluent, do not contain antimicrobial
properties against S. pneumoniae. This is in par with the studies done by Hindi, Al-mahdi, &
Chabuck, (2014) and Malu, Obochi, Tawo, & Nyong, (2009) which reported that the aqueous
extracts of ginger did not have any antimicrobial effect against streptococcus pneumonie. The
ineffectiveness of ginger to S. pneumoniae could be as a result of failure for the gimger
chemical components to destroy the cell wall of S. pneumoniae. Contrary, the S. aureus and S.
Pneumoniae are all gram positive bacteria, having the same cell wall structure (Hogg, 2005),
so the failure of ginger to have an effect on S. pneumoniae could be attributed to the resistance
of the bacteria.

The results of the study indicate no any zone of inhibition in their growth media in all the
concentrations and S. Pneumoniae was found to be resistant to the aquatic extracts. These
results are contradictory to the observations of Nader, Ghanima, Ali, & Azhar, (2010) who
prepared 20g of fresh ginger rhizomes, blended and soaked them in 100ml of hot water at 80℃
(shaker water bath) at 150 rpm for 24hrs. the resulted juice was extracted, air-dried and stored
at 4℃, reported that the aquatic extracts of ginger has the antimicrobial activity against the
streptococcus sp and the effectiveness decreased as the storage time of the extracts increases.
The effectiveness could be due to the better extraction method of letting the extracts to shake
for 24hrs. The difference in results may be due to the difference in the method used, the
resistance of the microorganisms used and the type of ginger used. Onianwah & Ho, (2018)
reported that storage temperature and time at which the samples are stored can affect the
effectiveness of the antimicrobial property of ginger. The difference in time were observed in
each and every hour and the effect of ginger were decreasing according to time. So the
difference might be due to the duration at which the extracts were stored. In this study, extracts
were stored for 72hrs.

20
The Microbial resistance of microorganisms in different geographical locations contributes
much to the difference in sensitivity of microorganisms to different antimicrobial agents. The
results of the studies that are done in different regions come up with different results. Romulo,
(2018) reported that the bioactive elements of medicinal plants differs according to the region,
however, the effect of water extracts of ginger on S. pneumoniae in vitro cannot be predicted
in this study.

5.1.3 MIC OF GINGER ON MICROORGANISMS

The results of this study showed that the zone of inhibition is found on S. aureus only with the
percentages of 80% and the diameter being 12mm and partially on 40% with the diameter of
9mm. there was no zone of inhibition in 20% and 10%. This implies that the minimum
inhibitory concentration for ginger on staphylococcus aureus is 40%, and a person who is
infected with the disease caused by staphylococcus aureus need to take more than 40g/100ml
of ginger extracts for its effectiveness. This differs with the study done by Rajsekhar,
Chandaker, & Upmanyu, (2012a) which reported that the MIC for ginger of staphylococcus
aureus was 20% (20g/100ml) with the diameter of 10mm. The study used one isolate and could
not be used to conclude the MIC of other strains of microorganisms, and the present study used
34 isolates. The difference in the results on MIC of ginger may be due to the difference in the
variety of ginger used and the resistance of the microorganisms.

The study did not find any zone of inhibition on streptococcus pneumoniae (figure 2). This
means that there is no MIC of ginger on streptococcus pneumoniae and no dose can be used
for patients suffering from diseases caused by the bacteria. This differs with the study by
Rajsekhar et al., (2012b) which found that the MIC was 20g/100ml with the diameter for zone
of inhibition of 20mm on streptococcus sp. the difference may be due to the resistance and the
variety of ginger used.

Different studies determined different MIC for different microorganisms. This may mean that
this study cannot conclude an exact MIC for ginger since it depends with the resistance of the
microorganism, the preparation of extracts and the type of ginger used.

5.2 CONCLUSION
The results of the study emphasize the usefulness of ginger in the treatment of diseases and the
need to enhance its exploitation on this regard. The concentrations of aqueous extract were

21
obtained and used in this study. The extracts exhibited an activity in Staphylococcus aureus
and no activity against Streptococcus pneumoniae. This justifies that the plant might be used
to treat infections that are caused by Staphylococcus aureus but not those infections caused by
Streptococcus pneumoniae. The extracts showed a larger Zone of inhibition on Staphylococcus
aureus at 80% (12mm) and thee diameter of the zone of inhibition decreased at 40% (9mm)
and no Zone of inhibition were exhibited at 20% and 10% as well as at streptococcus species.
This justifies that the amount of ginger, has an effect on an activity on Staphylococcus aureus
and the findings suggests that there is a potential to discover novel antibiotic agents from ginger
plant grown in Malawi.

5.3 RECOMMENDATIONS
After having the research findings using the methodology shown above, the following
recommendations were made.
➢ Ginger can be used as an alternative antibiotic to treat infections caused by
staphylococcus aureus with low severity.
➢ Ginger should not be used as an antibiotic to treat infections caused by Streptococcus
pneumoniae.

5.4 AREAS OF FURTHER STUDY

The aim of the study was to investigate the effectiveness of ginger as an antimicrobial agent
against Staphylococcus aureus and Streptococcus pneumoniae. However, further studies are
required to explore the effectiveness of ginger.
➢ Investigating the effectiveness of ginger using other solvents
➢ Investigating the effectiveness of ginger on other microorganisms that cause serious
diseases.
➢ Extracting and determining the antibiotic component of ginger to be used
commercially.
➢ Conduct clinical studies to determine the effect of more ginger consumption in the
body.

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nualReport_2017.pdf?ua=1.

Williams, P. C. M., Isaacs, D., & Berkley, J. A. (2018). Antimicrobial resistance among
children in sub-Saharan Africa. The Lancet. Infectious Diseases, 18(2), e33–e44.
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Woolhouse, M., Waugh, C., Perry, M. R., & Nair, H. (2016). Global disease burden due to
antibiotic resistance – state of the evidence, 6(1), 1–5.
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Lalitha, D. M. (2004, September 21). Manual on Antimicrobial Susceptibility Testing.

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Tripathi, P., Singh, A., Srivastava, A., Rekhi, L., & Ali, S. M. (2016). Antibacterial activity of
six indigenous Indian plants: Acacia nilotica (Fabaceae), Albizia saman (Fabaceae),
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APPENDICES

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APPENDIX A: PREPARATION OF MEDIA
Blood agar (BA)
1. Prepare the agar medium as instructed by the manufacturer. Sterilize by autoclaving
at 121oC for 15 minutes. Transfer to a 50oC water bath.
2. When the agar has cooled to 50 0C, add aseptically the sterile blood and mix gently
but well. Avoid forming air bubbles.
NB: The blood must be allowed to warm to room temperature before being added to the
molten agar.
3. Dispense aseptically in 15 ml amounts in sterile petri dishes
4. Date the medium and give it a batch number.
5. Store the plates at 2–8 0, preferably in sealed plastic bags to prevent loss of moisture.

Chocolate blood agar

1. Prepare as described for blood agar except after adding the blood, heat the medium in
a 70 0C water bath until it becomes brown in colour. This takes about 10–15 minutes
during which time the medium should be mixed gently several times.
2. Allow the medium to cool to about 45 0C, remix and dispense in sterile petri dishes as
described for blood agar.
NB: Care must be taken not to overheat or prolong the heating of the medium because this
will cause it to become granular and unfit for use.
3. Date the medium and give it a batch number. Store the plates as described for blood
agar.

Mueller Hinton agar

1. Prepare and sterilize the medium as instructed by the manufacturer. The pH of the
medium should be 7.2–7.4.
2. Pour into 90 mm diameter sterile petri dishes to a depth of 4 mm (about 25 ml per
plate). Care must be taken to pour the plates on a level surface so that the depth of the
medium is uniform.
Note: If the medium is too thin the inhibition zones will be falsely large and if too thick the
zones will be falsely small.

APPENDIX B: DATA COLLECTION TOOL

28
MICROBIOLOGY LABORATORY REQUEST FORM
Instruction: Please fill in the columns fully.
SAMPLE ID: Age: Sex:
M F
Microorganism: Date of subculture:
Time of subculture:
Clinical information/History: Antibiotic history (recent/current):

Test Required: CULTURE & SENSITIVITY

For microbiology department only. Do not write or tick below.

CULTURE

SENSITIVITY
ANTIBIOTICS Code S I R
Amoxicillin-Clavulanic acid AMC
Gentamycin GM
80% concentration of Ginger G1

40% concentration of Ginger G2

20% concentration of Ginger G3

10% concentration of Ginger G4

COMMENT:

Performed by:…………. Date performed: ……. /…… / 2019 Lab number:

Reviewed by: ………….. Date Reviewed: …….. /……. / 2019
Hint: S= sensitive, I = intermediate, R= Resistance
Created by: Shadreck Phiri

APPENDIX C: LETTERS

29
REQUEST LETTER FOR PERMISSION
Mzuzu University
Private bag 201
Luwinga
Mzuzu 2.

4th march, 2019.

The Research Committee

Mzuzu Central Hospital
Private Bag 209
Mzuzu

Dear Sir/Madam
REQUEST FOR PERMISSION TO CONDUCT A RESEARCH STUDY AT MZUZU
CENTRAL HOSPITAL

I am a fourth year student pursuing a Bachelor of Science in Biomedical Sciences at the

above mentioned university. In partial fulfilment for the award of the degree, I am supposed
to conduct a research study which I intend to carry out at Mzuzu Central Hospital. The topic
of my study is; “investigating the effectiveness of zingiber officinale (ginger) as an
antibiotic agent against staphylococcus aureus and streptococcus pneumoniae”.

The study findings and recommendations will offer an insight and be much beneficial to the
public should they be carefully considered. My scheduled time to collect the data for the
study is within the months of March and April 2018.
I am looking forward to your favourable response.
Sincerely yours,

Shadreck phiri Master chisale

(Researcher) (supervisor)

APPENDIX D: METHOD FOR SUSCEPTIBILITY TESTING

Method

30
 1. Using a sterile wire loop, touch 3–5 well-isolated colonies of similar appearance to
the test organism and emulsify in 3–4 ml of sterile physiological saline or nutrient
broth.
2. In a good light match the turbidity of the suspension to the turbidity standard (mix the
standard immediately before use). When comparing turbidities it is easier to view
against a printed card or sheet of paper.
3. Using a sterile swab, inoculate a plate of Mueller Hinton agar. Remove excess fluid
by pressing and rotating the swab against the side of the tube above the level of the
suspension. Streak the swab evenly over the surface of the medium in three directions,
rotating the plate approximately 60o to ensure even distribution
4. With the petri dish lid in place, allow 3–5 minutes (no longer than 15 minutes) for the
surface of the agar to dry.
5. Using sterile forceps, needle mounted in a holder, or a multidisc dispenser, place the
appropriate antimicrobial discs, evenly distributed on the inoculated plate. ensure the
discs are correctly placed.
Note: The discs should be about 15 mm from the edge of the plate and no closer than about
25 mm from disc to disc. No more than 6 discs should be applied (90 mm dish). Each
disc should be lightly pressed down to ensure its contact with the agar. It should not
be moved once in place.
6. Within 30 minutes of applying the discs, invert
the plate and incubate it aerobically at 35 0C for 16–18 h (temperatures over 350C
invalidate results for oxacillin).
7. After overnight incubation, examine the control and test plates to ensure the growth
is confluent or near confluent. Using a ruler on the underside of the plate measure the
diameter of each zone of inhibition in mm. The endpoint of inhibition is where growth
starts.

31