This document summarizes a research study that investigated the antimicrobial activity of lemon juice and vinegar on pyelonephritis-causing bacteria. The study found that lemon juice and vinegar were effective at inhibiting the growth of E. coli, Klebsiella pneumoniae, and Proteus mirabilis bacteria. Lemon juice produced inhibition zones of 0.099, 0.069, and 0.102 for the three bacteria, respectively. Vinegar also showed inhibition zones ranging from 0.088 to 0.111. The antimicrobial effects are attributed to organic acids like citric acid in lemon juice and acetic acid in vinegar, which disrupt the bacterial cell membrane and intracellular processes. While cranberry juice did not inhibit

1. ANTIMICROBIALACTIVITY OF
LEMON JUICE AND VINEGAR ON
PYELONEPHRITIS (KIDNEY
INFECTION) DISEASE CAUSING E.
COLI, KLEBSIELLA PNEUMONIA,
PROTEUS MIRABILIS BACTERIA

2. Chapter 1: Introduction
Introduction
• Pyelonephritis can be seen as a type of urinary tract
infection caused by bacterial invasion in which one or
both kidneys in the human body become infected.
• The information begins in the urethra for the bladder and
after some time it travels to the kidneys spreading the
infection (Okeke et al. 2019).
• In the current medical practices, Outpatient oral antibiotic
therapy (OOAT) with the Fluoroquinolone has been
observed to be successful for the patient with mild
uncomplicated pyelonephritis (Padron, 2020).
• However, treatment with drugs can cure this type of
infection in 2 to 3 days, but the medication must be
continued for the entire period of prescription which is
usually fourteen days (Hindi et al. 2018).

3. Research rationale
• As per the information obtained from ncbi.nlm.nih.gov
(2021), acute pyelonephritis has been observed to be a
severe urinary tract infection (UTI) that has been estimated
to occur around 11 cases for 10,000 inhabitants.
• At the same time, these diseases have been observed to be
4 to 5 times more frequent in the female body in
comparison to male individuals.
• It has also been observed that 1% of boys and 3% of girls
have acute pyelonephritis at the age of 7 and these disease
development incidents are highest among the women
within the age group of 15 to 29 (patient.info, 2020).
• In addition to that, the antibiotics used to treat this disease
condition include fluoroquinolone, penicillin, amoxicillin-
clavulanate potassium, cephalosporins, and trimethoprim-
sulfamethoxazole.
• However, these antibiotic agents have the most common
frequent side effects including gastrointestinal reactions
that cause nausea, dyspepsia, and vomiting.
• At the same time, CNS reactions including dizziness,
insomnia, headache, and other kinds of irritations such as
allergic reactions have also been observed in many cases
(Kalia et al. 2020).

4. Aim and Objectives
Aim:
• The main aim of this research is to investigate the antimicrobial activity of lime juice and
vinegar on pyelonephritis disease caused by bacterial growth of E. Coli, Klebsiella
Pneumonia, Proteus Mirabilis Bacteria.
Objectives:
The objectives of this research are,
• To investigate the current scenario of pyelonephritis disease and its treatment options
• To analyze the alternative ways for treating pyelonephritis
• To implement natural compounds with antimicrobial activity
• To evaluate the results of antimicrobial activity shown by the natural compounds against
the development of pyelonephritis

5. Literature Review
• Antimicrobial activity of apple cider vinegar
against Escherichia coli, Staphylococcus
aureus, and Candida albicans
• Antibacterial activity of Citrus lemon fruit
juice extract
• Efficacy of vinegar, salt solution, and lemon
juice as antimicrobial agents in reducing the
microbial load

6. Chapter 2: Materials and methods
Materials Used
• This research was conducted with the primary
methodologies in which the researcher has
performed their own experiments and
laboratory procedures.
• In this research, the materials that have been
used include cranberry, lemon juice,
tomatoes, vinegar, oranges brought from the
enbus supermarket store.
• The materials which have been brought have
been listed before the initiation of this
research work and each of the materials has
been added to the list based on the research
requirements (Madarisa, 2019)
• Other materials that have been included in
this research work are Benson Burner,
bacterial culture, thermometer, agar plate,
Gentamicin antibiotic plate.
• Some more materials have also been included
in this research that are micro tips, bickers,
falcon tubes, watch glasses, forceps, glass
rods, some chemical solutions, pH controlling
devices, and stains.

7. Sample collection
• The samples of Cranberry, vinegar, lemon
juice were collected from the supermarket
and opened in the laboratory in the
sterilized conditions between Benson
burners
• Primary sterilization of the surface of the
tomatoes and oranges was done with 70%
alcohol.
• The bacterial culture was collected from
the laboratory and where stored in a cold
storage for father experimentation (Liu
and Hannig, 2020).
• The collection of materials work done on
the basis of the list that has been made
before the start of the experiment and
research procedure.

8. Bacterial culture preparation
• Preparation of bacterial culture is one of the essential
tasks which is common to every microbiology
experiment in which the bacterial population is
allowed to grow in the presence of the essential
nutrition.
• The microorganisms that grow in a culture medium
and search for others must be kept under an
incubator (Liu et al. 2018).
• In the absence of high-quality media the possibility
of cultivating accurate reproducible as well as
repeatable microbiological test results can be
minimized due to bacterial contamination (Salari et
al. 2019).
• In this experiment, both the culture of E. coli,
Klebsiella pneumonia, and Proteus mirabilis were
shrunk under a nutrient agar plate and were
incubated at 37 degree Celsius for a day.
• After 24 hours, the culture was picked up and
isolated in the bacterial community with specific
characteristics identification (Khan et al. 2018).
• The density of each culture suspension was checked
afterwards and the normal range of density was
considered to be 0.08 >> 0.1 at 600nm.

9. To check antimicrobial activity
Well diffusion method
• Well diffusion method can be seen to be a widely used
methodology to evaluate antimicrobial activity of
plants and the microbial extract.
• The principle of this well diffusion method described
the antibiotic-impregnated disk, pleased on the agar
that was previously isolated with the bacteria of
interest and picking up the monsters (Missoun et al.
2019).
• In this experimental study, the materials that have been
used for this well diffusion method are N. Agar plate,
bacterial suspension, and Swab
• Micropipette and Micro tips have been included in this
research for or the delivery of fruit juice and vinegar in
the culture (Guo et al. 2019).
• Three major parts of checking antimicrobial activity
were taken under consideration after 1 day of
incubation for each of the bacterial cultures.

10. To check antimicrobial activity (Continue)
Disk diffusion method
• Disc diffusion method (DDM) is another well used agar
diffusion method (ADM), in which antibiotic extracts are tested
with the diffusion from its reservoir through the agar medium
seeded with microorganism.
• The main purpose of this disc diffusion susceptibility method is
to determine the resistance or sensitivity of particular bacteria
(pathogenic aerobic and facultative anaerobic) against different
antimicrobial compounds (Chalad et al. 2018).
• In this experimental study of disk diffusion, the materials that
have been used for this well diffusion method are N. Agar
plate, bacterial suspension, and Swab.
• , a drop of each of the natural compounds (in cranberry, lemon
juice, tomatoes, vinegar, orange) was taken in the experimental
plate made up of filter paper which was 6 mm in diameter.
• For each of the bacterial stains, different plates have been
prepared and after adding the bacterial culture the Petri dishes
taken for incubation at 37 degree Celsius for 24 hours.
• After being fusion for one day, the bacterial cultures were taken
for observation where three major observations have been
considered to be made (Gefen et al. 2017).

11. Chapter 3: Result and Discussion
Results
• Based on the observations, this research has come to the
results of bacterial inhibition in response to different natural
antibiotics such as cranberry, lemon juice, tomatoes, vinegar,
and orange.
Result of cranberry and lemon juice:
• Applying the cranberry and lemon juice it has been found
that the bacterial innovation was occurred in the lime juice
but not in cranberry juice.
• The result has found that the inhibition zone of E. coli was
0.099 and in case of Klebsiella pneumoniae it was 0.069.
• At the same time, Proteus mirabilis has shown the result of
0.102 which is the highest measurement of innovation zone
after applying the lemon juice.
E. coli Klebsiella
pneumoniae
Proteus mirabilis
Lemon Juice
0.099 0.069 0.102
Cranberry Juice
_ _ _

12. Results (Continue)
Result of tomatoes, vinegar and orange:
• After taking the observations of lemon juice and
cranberry extract, the researchers have also used
tomatoes, vinegar, and orange, and innovation
has been found.
• In the presence of vinegar, E. coli has shown a
zone of inhibition of 0.111, whereas Klebsiella
pneumoniae has shown a zone of inhibition of
0.099.
• Moreover, Proteus mirabilis has also shown an
innovation in the presence of vinegar and an
inhibition zone of 0.088 has been observed.
E. coli Klebsiella pneumoniae Proteus mirabilis
Vinegar 0.111 0.099 0.088
Orange Juice
Tomatoes

13. Discussion
• Bacterial growth can be restricted with the natural
antimicrobial agents and this has been supported with
the data findings in this research.
• Based on the result and the data findings it has been
observed that the bacterial propagation and their
growth can be restricted with the help of
incrementing some natural compounds such as
vinegar, and lemon juice.
• At the same time, bacterial growth restriction or
inhibition has also been observed with the application
of orange juice, and tomato (Hase et al. 2018).
• Therefore, it can be stated that there is a significant
antimicrobial activity of lime juice and vinegar on
pyelonephritis disease caused by bacterial growth of
E. Coli, Klebsiella Pneumonia, Proteus Mirabilis
Bacteria (Singh et al. 2017).
• bacterial growth has been effectively restricted as
well as inhibited with the application of these natural
compounds (Aljerf and AlMasri, 2018).

14. Discussion (Continue)
• The reason behind the bacterial growth inhibition
has also been understood as the antimicrobial
activity of organic acids present in these natural
compounds.
• The presence of acetic acid (CH₃COOH) in
vinegar, citric acid (C₆H₈O₇) in lemon juice and
orange as well as the presence of malic acid
(C4H6O5) in tomatoes has effectively inhibited the
bacterial growth (Roy et al. 2018).
• The organic acid spreads through the bacterial cell
membrane and separates into anions (-ve) and
protons/cations (+ve) (Aras et al. 2018).
• Eventually, these two negatively and positively
charged ions inhibit the electronic balance within
the bacterial cells and develop suppression in the
action of cellular enzymes as well as the
transportation system in the bacterial cell (Di
Rienzi et al. 2018).

15. The success of the research study
• There were two major hypotheses in this study that were
taken for justification and after the observations and data
findings, it can be stated that the null hypothesis is
rejected.
• According to the alternative hypothesis, there is a
significant antimicrobial activity of lime juice and
vinegar on pyelonephritis disease caused by bacterial
growth of E. Coli, Klebsiella Pneumonia, Proteus
Mirabilis Bacteria (Aras et al. 2018).
• The main reason behind it has also been identified as the
presence of organic acids in these natural compounds.
• However, due to the presence of low amounts of organic
acids in cranberry, research has shown a negative result
with the application of cranberry juice.

16. Comparing the results with previous
studies
• The result of this research study has also when
compared with the previous studies in this area and a
significant similarity has been found.
• Comparing the results of this experiment with the
previous studies has not shown any significant
difference between them and the minimum
difference has occurred by chance (Andrés-Barrao
and Barja, 2017).
• The difference of place of experiment, bacterial
strain variance, dissimilarity in the experimental
procedure, and other factors are responsible for the
dissimilarities which are not significant in the 5%
significance level and 95% confidence level (Fan et
al. 2017).
• Lastly, it can also be stated that this study has added
some significant knowledge in this field by virtue of
comparing the response of different types of
bacterial strain in different natural compound
applications (De Filippis et al. 2018).

17. Explaining the refutation in some
areas
• The overall study was significantly successful
and the desired results have been found for
each of the bacterial strains in different
natural compound applications.
• However, in some cases, refutations have also
been observed including the negative result of
cranberry juice in inhibiting the bacterial
growth (Villegas-Guzman et al. 2017).
• The refutation in the cranberry juice
experiment can be justified with the
description of limited organic acid presence
in cranberry (Andrés-Barrao and Barja,
2017).
• These insufficient organic acids were not
capable of controlling the bacterial growth
and restrict them to propagate.

18. Alteration of methods for providing
more definitive results
• This study has provided successful results regarding
the bacterial growth innovation in presence of different
natural antibiotics.
• However, some alterations in the experimental
procedure in future studies can improve the delivery of
the overall research.
• In this regard, the consideration of moisture, pH levels,
warmth, and oxygen level is required which are the big
4 physical as well as chemical factors affecting the
growth of microorganisms (Di Rienzi et al. 2018).
• At the same time, the control of microorganisms with
some mythological implementations could have also
improved the result of these experiments.
• The control of temperature, osmotic pressure,
desiccation, radiation, and filtration could have
enhanced the overall experiment while providing
better results.

19. Chapter 4: Conclusion
Conclusion
• Based on the analysis it can be concluded that
Pyelonephritis causing bacteria including E. coli
Klebsiella pneumoniae, and Proteus mirabilis shows
a significant innovation in presence of organic acids.
• This experiment has also revealed that the amount of
organic acids present in a natural compound is
important and there is a threshold level that needs to
be crossed for restricting the bacterial growth.
• This research was based on the investigation of
antimicrobial properties present in the natural
compounds such as lemon juice, vinegar, oranges,
tomatoes, and cranberry.
• Among these natural sources of organic acids, all of
the compounds have shown positive results against
the bacterial growth except cranberry.

20. Research limitations
• This research has provided significant results
regarding the antimicrobial capability of natural
compounds that restrict the growth of bacteria
causing urinary tract infections in the human body.
• However, the insufficiency of data available on the
internet on several topics has developed some
limitations in this research.
• The unavailability of sufficient primary researches in
this area has also developed some serious constraints
of knowledge that were required for the effective
delivery of this research.
• The effects of several factors including pH level,
temperature, oxygen level, and others were not
considered in this research which could have
significant effects on the result.

21. Chapter 5: Future scope
Scope in the research field:
• The study will provide a template for future studies in
the same field by virtue of providing the data support
and help the future researchers by providing
experimental strategies.
Scope in medical advancement:
• The present treatment options for Pyelonephritis are
the chemical antibiotics which have a wide range of
side effects in the human body.
Scope in genetic engineering:
• The development of antibiotic resistance among the
bacteria is due to the mutation in their genes and the
development of antibiotic resistance genes.
Scope in bacterial meta-population analysis:
• This study has not only investigated the impacts of
different natural compounds on a single bacterial
growth but also has provided suggestions regarding
three different bacterial strains.
• Lastly, it can be stated that this study has different
future scopes in various fields of research as well as
practical implementations.
Scope in the research field
Scope in medical advancement
Scope in genetic engineering
Scope in bacterial meta-
population analysis

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