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Antibacterial Activity of Herbal Plants Popularly Used in India (1)

The document discusses the prevalence and impact of urinary tract infections (UTIs), particularly among women and the elderly, highlighting the increasing antibiotic resistance of uropathogens. It explores the potential of herbal plants, such as Ficus bengalensis, Salvadora persica, and Vitex negundo, as alternative treatments against UTIs due to their antibacterial properties. The research aims to isolate bacteria from UTI samples, conduct biochemical tests, and screen natural remedies for efficacy against these infections.

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0% found this document useful (0 votes)
3 views67 pages

Antibacterial Activity of Herbal Plants Popularly Used in India (1)

The document discusses the prevalence and impact of urinary tract infections (UTIs), particularly among women and the elderly, highlighting the increasing antibiotic resistance of uropathogens. It explores the potential of herbal plants, such as Ficus bengalensis, Salvadora persica, and Vitex negundo, as alternative treatments against UTIs due to their antibacterial properties. The research aims to isolate bacteria from UTI samples, conduct biochemical tests, and screen natural remedies for efficacy against these infections.

Uploaded by

Rohit 89
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Antibacterial Activity of Herbal Plants Popularly Used in

India Against Urinary Tract Infection


Chapter I
Introduction:
Urinary tract infections (UTIs) are very common bacterial infections in women and older people.
They can be quite uncomfortable for patients, although they are usually not life-threatening.
However, they do cause a lot of distress. UTIs also cost a lot of money for healthcare and society.
In the USA alone, UTIs lead to 7 million visits to the doctor's office every year (Shireen, 2011).
Except among infants and the elderly, the infection occurs more commonly in women than in men
and it was estimated that about 40–50% of women experience one episode in their lives and 20–
30% of them have other episodes. For women between 1 year and up to 50 years. (Kasperet.al,
2018)

In individuals without anatomical or functional abnormalities, UTIs are generally self


limiting, but have a propensity to recur. Uropathogens have specialized characteristics, such as
the production of adhesins, siderophores and toxins that enable them to colonize and invade the
urinary tract, and are transmitted between individuals both through person-to-person contact and
possibly via food or water. Although generally self-limiting, treatment of UTIs with antibiotics
leads to a more rapid resolution of symptoms and is more likely to clear bacteriuria, but also Rubin
et.al, 1992reported that resistant uropathogens and commensal bacteria adversely affects the gut
and vaginal microbiota.

Ronald, 2001 noted that the pathogens traditionally associated with UTI are changing
many of their features, particularly because of antimicrobial resistance. The etiology of UTI is
also affected by underlying host factors that complicate UTI, such as age, diabetes, spinal cord
injury, or catheterization. Consequently, complicated UTI has a more diverse etiology than
uncomplicated UTI, and organisms that rarely cause disease in healthy patients can cause
significant disease in hosts with anatomic, metabolic, or immunologic underlying disease.

Hootan, 1999 investigated the microbial etiology of urinary infections has been regarded
as well-established and reasonably consistent. Escherichia coli remains the predominant
uropathogen (80%) isolated in acute community-acquired uncomplicated infections, followed by
Staphylococcus saprophyticus (10% to 15%). Klebsiella, Enterobacter, and Proteus species, and
enterococci infrequently cause uncomplicated cystitis and pyelonephritis.

The majority of community-acquired symptomatic UTIs in elderly women are caused by


E coli. However, gram-positive organisms are common, and polymicrobial infections account for
up to 1 in 3 infections in the elderly. In comparison, the most common organisms isolated in
children with uncomplicated UT1 are Enterobacteriaceae. Etiologic pathogens associated with
UT1 among patients with diabetes include Klebsiella spp., Group B streptococci, and
Enterococcus spp., as well as E coli. Patients with spinal cord injuries commonly have E coli
infections. Other common uropathogens include Pseudomonas and Proteus mirabilis(Stamm
et.al., 1993).

As uropathogens are increasingly becoming resistant to currently available antibiotics, it


may be time to explore alternative strategies for managing UTI(Warren et al., 1999).

In the last decades, the extensive use of antibiotics has resulted in the emergence of
antibiotic-resistant bacterial pathogens and leads to the spread of antibiotic resistance. (Khameneh
et.al.,2019). Additionally, because of the chronic nature of UTIs and the potential for antibiotic
resistance, a promising approach to prevention and treatment is favourable. These days various
approaches have been developed to overcome the problems associated with antibiotic resistance.
Complementary and alternative medicine (CAM) has been recognized as an effective approach
for the treatment of infection by antibiotic-resistant bacteria(Khamenehet.al.,2019).

Clinically research suggests the best natural options for long-term prevention include
probiotics, medical herbs, vitamins, and elements that have also been shown to prevent
UTIs(Poulios et.al., 2020). So, we could hope that using CAM in the treatment of UTI could
provide desirable results, especially when combined with a routine antibiotic regimen.

According to Anjana et.al., 2009 plants generate a diverse range of secondary metabolites
that are utilized in the pharmaceutical industry as lead compounds or as direct precursors. It is
anticipated that plant extracts exhibiting target sites that differ from those employed by antibiotics
will exhibit efficacy against drug-resistant microbial pathogens.

Some of the herbal plants selected to test the antibacterial activity against UTI causing
bacteria in this research are Ficus bengalensis (Banyan tree), Salvadorapersica(Miswak),
Vitexnegundo(Nirgundi).

Salvadorapersicais an evergreen small tree belonging to family Salvadoraceae, commonly known


as 'Pilu', 'Jal' and 'Toothbrush tree' and is widely distributed in India, Africa, Saudi Arabia, Iran,
Israel and Pakistan. It has been claimed in traditional literature to be valuable against a wide
variety of diseases(U. Bhadoriya et.al., 2010).

Miswak plant has flavonoids, sterols, glycosides, terpenes, carbohydrates, alkaloids as its
secondary metabolites which are investigated to be beneficial in treating the Urinary Tract
Infections although there are no such research being performed to test the efficacy of the miswak
plant against uti.
From the family of Lamiaceae, Vitexnegundois a large, deciduous, aromatic shrub or small,
slender tree upto 4.5 m tall. it is found throughout India and Sri Lanka. It is a commonly available
medicinal plant in India and it is also known as the "sarvarognivarini"- remedy for all diseases.
Therefore, it is used to treat many diseases due its antioxidant, antitumour, hepatoprotective and
antimicrobial activities (Joshi et.al., 2018).

The secondary metabolites of nirgundi consists of iridoids, diterpenoids, ecdysteroides,


flavonoides which are been reported to have antibacterial activities against the bacteria isolated
from the urinary tract infections.

Ficus have been known for their vast number of species, consisting of more than 800 species in
the form of trees, vines, shrubs, epiphytes, and hemiphytes. Ficus genera belong to the Moraceae
family of Urticales order under the classification of Dicotyledone and Spermatophyte phylum of
the Plantae kingdom. There are more than 800 species of Ficus that have been discovered. Ficus
plants are generally known as figs or fig trees. The genus is distributed in various regions across
the tropical and sub-tropical areas, mainly in Asia, America, Australia, and Africa (S. Murugesu
et.al., 2021).

Presence of few secondary metabolites such as flavonoids, alkaloids, glycosides, phenolic


compounds has been screened out from the different parts of the plants such as bark, leaves, roots,
stems etc.

In recent years, emerging antimicrobial resistance against common uropathogens has become a
major challenge for management of UTI. This problem is increasing in the countries which
economically weak or are developing. Antimicrobial susceptibility pattern should be monitored
regularly to select the choice of treatment modality. Thus to overcome this emerging problem new
choice of treatment should be implied such as use of herbal medicines and regular study of the
antimicrobial resistance pattern should be done.

Extraction of medicinal plants is a process of separating active plant materials or


secondary metabolites such as alkaloids, flavonoids, terpenes, saponins, steroids, and glycosides
from inert or inactive material using an appropriate solvent and standard extraction procedure.
Plant materials with high content of phenolic compounds and flavonoids were found to possess
antioxidant properties.

Several methods were used in the extraction of the medicinal plants such as maceration,
percolation, infusion, decoction, digestion and Soxhlet extraction, microwave assisted extraction,
ultrasound extraction. In addition, Thin-Layer Chromatography (TLC), Paper Chromatography
(PC), and Gas Chromatography (GC) were used in separation, and purification of the secondary
metabolites.
The solvents used for the extraction of the medicinal plants is known as the menstruum.
The choice of solvent depends on the type of plant, part of plant to be extracted, nature of the
bioactive compounds, and the availability of solvent. In general, polar solvents such as water,
methanol, and ethanol are used in the extraction of the polar compounds, whereas non-polar
solvents such as hexane and dichloromethane are used in the extraction of the non-polar
compounds.

This research was undertaken with the following objectives:

i) Isolation of bacteria from UTI infected samples.


ii) Biochemical tests of isolated bacteria.
iii) Screening of the natural remedies on the bacteria from the urinary tract infection.
Chapter II
Review of Literature:
Urinary tract infections (UTIs) are still among the most prevalent bacterial illnesses worldwide,
having been first reported in Egypt around 1550 BC. (Nickel, 2005). The prevalence of UTI
seems to be a J-shaped distribution, with higher frequency among very young children which
gradually increases with age (Johnson, 1991).
An estimated 35% of healthy women experience urinary tract infection symptoms at
some point in their lives. Each year, dysuria, or painful and frequent urination, affects about
5% of women (Hootan, 2003). Worldwide, approximately 150 million people are diagnosed
with UTIs, causing a heavy burden on the health financial system with around 6 billion dollars
in healthcare spending each year (Foxman, 2002).
Due to prolapse and urinary retention, women's shorter urethral length as shown in
(figure 1), frequent vaginal colonization, and obstructions to urine flow and full bladder
emptying, they are particularly vulnerable to infection. (Rowe, 2013).

Fig: 1.Difference In length of urinary bladder of male and female.

UTIs are classified as either uncomplicated or complicated. Individuals with


uncomplicated UTIs usually have no structural or neurological urinary tract abnormalities and
are otherwise healthy. (Hootan 2012), these infections are differentiated into lower UTIs
(cystitis) and upper UTIs (pyelonephritis) (Hannan, 2012). Several risk factors are associated
with cystitis, including female gender a prior UTI, sexual activity, vaginal infection, diabetes,
obesity and genetic susceptibility (Foxman 2014),

UTIs classified as complicated are those linked to conditions that impair the urinary
tract or the immune system, such as urinary obstruction, immunosuppression, pregnancy, renal
failure, renal transplantation, urinary retention due to neurological diseases, and the presence
of foreign bodies like calculi, indwelling catheters, or other drainage devices. (Lichtenberger,
2008). In the United States, 70-80% of complicated UTI are attributable to indwelling catheters
(Lo, 2014), accounting for 1 million cases per year.

CAUTIs, or catheter-associated urinary tract infections, are the most common cause of
subsequent bloodstream infections and are linked to higher rates of morbidity and mortality.
Diabetes, older age, gender, and prolonged catheterization are risk factors for acquiring a
CAUTI. (Chenoweth, 2014).

UTIs are caused by both Gram-negative and Gram- positive bacteria, as well as by
certain fungi. The most common causative agent for both uncomplicated and complicated UTIs
is uropathogenic Escherichia coli (UPEC).

For the agents involved in uncomplicated UTIs, UPEC is followed in prevalence by


Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, group B
Streptococcus (GBS), Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and
Candida spp (Kline, 2011).

For complicated UTIs, the order of prevalence for causative agents, following UPEC
as most common, is Enterococcus spp., K. pneumoniae, Candida spp., S. aureus, P. mirabilis,

P. aeruginosa (Levison, 2013).

In healthy persons, urine was regarded as sterile to microorganisms. Because of their


high urea concentrations, comparatively low pH (6.0 on average), and increased salinity, their
composition is not conducive to the growth of microorganisms (Aragon, 2018). Certain uro-
pathogenic bacteria are inhibited from growing even by nitrates in a slightly acidic
environment, although certain proteins also possess antibacterial properties. Not only that, but
constant urine elimination stunts the growth of microorganisms.

Fimbrial adhesions are expressed by the uropathogenic bacteria and are attached to the
glycolipids and glycoproteins on the surface of the epithelium as shown in (figure 2). Bacteria
are able to persist in the urinary system and withstand urine flow in this way. In addition, the
bacteria release toxins,haemolysin, and colony necrotizing factors. These agents compromise
the integrity of the epithelium, allow bacteria to invade, and raise the risk of infection (Behzadi
2019). Uropathogens have the ability to proliferate and internalize into host epithelial cells,
creating areservoir for recurrent infection (Sheerin, 2019).
Fig: 2. Pathogenesis of urinary tract infection.

Antibiotic susceptibility testing should be the basis for treatment decisions because uro-
pathogens have the potential to develop multidrug resistance. Greater than or equal to 100,000
CFU/mL is considered a diagnostic threshold for urinary tract infections (UTIs), yet this
threshold frequently yields false negative results, meaning that many pertinent illnesses go
undetected (Little, 2006).

One of the biggest concerns for world health today is antibiotic resistance. Bacteria
resistant to several drugs are evolving and proliferating quickly. endangering our capacity to
treat widespread infectious illnesses. Presumably, one of the primary causes of drug resistance
is the indiscriminate use of commercial antibiotics. The World Health Organization (WHO)
estimates that by 2050, drug-resistant illnesses might claim 10 million lives annually and
seriously harm the world economy. Finding goods with antimicrobial qualities that could be
used to create innovative and effective antibiotics is therefore vital.

Although UTIs are frequently treated with current antibiotics, interest in investigating
alternative, natural therapies has surged due to the emergence of antibiotic-resistant bacteria
and the possibility of unfavorable side effects.

Since ancient times, people have used plants as medicine, and this tradition is still in
use today. Research indicates that around 80% of people living in poor nations take medications
made from plants (Nirmal, 2013). Since medications made from plants are safer than those
made from synthetic materials (Nisar, 2018), the future will see an increase in their usage. It is
projected that the global commerce in medical plants and their products will be worth USD 5
trillion by 2050 (Zahra, 2020).

Many plant components, including the leaf, root, flower, fruit, peel, bark, seed, stem,
and rhizome, among others, have a high concentration of bioactive chemicals that have the
potential to be used medicinally (Ifesan, 2013).

Salvadora persica is a little tree or shrub that rarely grows larger than one foot in
diameter. It has a crooked trunk. Its pale bark features pendulous extremities and is scabrous
and broken. The tree's inner surfaces are an even paler shade of brown, and the root bark
resembles sand. It tastes warm and spicy and has a nice scent. Young branches have grayish
brown bark on the main stem and mild color elsewhere. They are green in color with slightly
rough bark. The leaves are 3*7 cm and are light to dark green in color, oblong-elliptic to almost
round. They occasionally have dense, rather loose hairs and glandular spots that resemble
warts. They are fairly meaty (Khatak, et.al., 2010).

S. persica is a widely used chewing stick (Miswak) in the Muslim world at large as well
as the Indian subcontinent (Soliman, 2007). The plant Salvadora persica, which is a member of
the Salvadoraceae family, contains carbohydrates, alkaloids (salvadorine), steroids, terpenoids,
saponins, flavonoids (kaempferol and quercetin), and glycosides (kaempferol 3-L
rhamnosyl‑7‑xylopyranoside) (Kamil, 2002).

The use of plant extracts against pathogenic bacteria—which are often linked to
antibiotic-resistant illnesses in hospital settings—has been the subject of several investigations
(Condo, et.al., 2020). Furthermore, S. persica is well known for its pharmacological properties,
and this plant has been linked to a wide range of biological activities, including antitumor,
hypoglycemic, antibacterial in vitro and in vivo, anticonvulsant, anti-inflammatory,
antioxidant, analgesic, antiulcer, sedative activity, and enzyme inhibitory effects (Chelli-
Chentouf et.al., 2022; Aumeeruddy et.al., 2018).

Spread across India, Vitex negundo Linn. is a fragrant shrub in the Verbenaceae family.
It is the preferred medication in the Ayurvedic medical system for treating pain, inflammation,
and other related conditions. There have been reports of strong analgesic and anticonvulsant
properties in leaves. Leaves are rich in alkaloids, glycosidic iridoids, terpenoids, and
polyphenolic chemicals. (Nagareskar et.al., 2010).
It is well known that medicinal plants can treat a wide range of illnesses. One of the ten
herbal remedies that the DOH has recognized as having therapeutic value and efficacy is V.
negundo Linn (Rana, 2014). Additionally, a study found that several compounds isolated from
V. negundo Linn may have analgesic, antifungal, and antibacterial qualities (Pawar, et.al.,
2017).

Certain species are revered in India. For example, Ficus benghalensis, also known as
the banyan tree and a symbol of spiritual wisdom and everlasting life, is known as India's
National Tree. (Gopukumar, et.al., 2015). Ficus species is one of the largest genera of plant
kingdom with promising phytoconstituents from various classes of compounds including,
phenols, flavonoids, sterols, alkaloids, tannins, saponins, terpenoids etc (Rao et.al., 2014). The
leaves and bark of F.benghalensis are rich in Flavonoids, phenols, terpenoids and terpenes
(Naquvi et.al., 2015).
To verify susceptibility to specific empirical antimicrobial drugs or identify resistance in
individual bacterial isolates, the clinical microbiology laboratory must undertake antimicrobial
susceptibility testing. In the case of Streptococcus pyogenes, which exhibits persistent
susceptibility to penicillin, empirical therapy remains effective against certain bacterial
infections due to the absence of resistance mechanisms. When dealing with species (such as
those in the Enterobacteriaceae, Pseudomonas, Staphylococcus, Enterococcus, and
Streptococcus pneumoniae families) that may have developed resistance mechanisms, it is
crucial to test the susceptibility of individual isolates (James et.al., 2009).

Antimicrobial susceptibility testing of noteworthy bacterial isolates is a crucial function


of the clinical microbiology laboratory. Testing aims to ensure susceptibility to preferred
medications for specific diseases and identify potential treatment resistance in common
organisms. The most popular testing techniques are rapid automated instrument procedures that
use commercially marketed materials and devices, or broth microdilution. The agar well
diffusion and disk diffusion manual methods are two that offer flexibility and potential cost
reductions.

Disk Diffusion Method: The disk diffusion susceptibility method has been well-
standardized and is straightforward and useful (Jorgensen et.al., 2007). The procedure involves
covering the surface of a sizable Mueller-Hinton agar plate (150 mm in diameter) with a
bacterial inoculum containing roughly 1-2 × 10 CFU/mL. Placed on the inoculated agar surface
are up to twelve commercially manufactured paper antibiotic disks with a predetermined
concentration. The plates are incubated at 35°C for 16–24 hours before the results are
determined. Every antibiotic disk has its growth inhibition zones measured to the closest
millimeter. The diameter of the zone reflects both the drug's rate of diffusion through the agar
media and the isolate's susceptibility as shown in (figure 5). The criteria listed in the US Food
and Drug Administration (FDA)-approved product inserts for the disks or those published by the
Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for
Clinical Laboratory Standards or NCCLS) are used to interpret the zone diameters of each drug.
The disk diffusion test yields "qualitative" results because it does not yield a minimum
inhibitory concentration (MIC), but rather a susceptibility category (i.e., susceptible,
intermediate, or resistant). However, by comparing zone diameters with standard curves of that
species and drug stored in an algorithm, some commercially accessible zone reader devices
claim to be able to generate an estimated MIC for certain organisms and antibiotics (Nijs et.al.,
2003).
Fig: 5. Disk-Diffusion Method
Well Diffusion Method: Diffusion method using Agar wells an extensively used
technique for assessing the antibacterial activity of plants or microbial extracts is the Agar well
diffusion method (Valgas et.al., 2007). The process of inoculating the agar plate surface
involves covering the entire surface with a volume of the microbial inoculum, much like in the
disk-diffusion approach. The antimicrobial agent or extract solution at the appropriate
concentration is then added to the well in a volume of 20–100 mL after an aseptic hole with a
diameter of 6–8 mm is punched using a sterile cork borer or a tip. Agar plates are then cultured
in the appropriate environment for the test microorganism. Inhibiting the growth of the tested
microbiological strain, the antimicrobial drug diffuses throughout the agar medium as shown
in (figure 6).

Fig: 6. Well-Diffusion method


Chapter III
Materials and methods:
Sample collection:
All samples were collected from different places, including some hospitals and laboratories. 94
samples were collected in sterile urine containers, kept in an icebox, and transferred to the
bacteriological laboratory at Department of microbiology, Mahatma Jyotiba Phule Rohilkhand
University, Bareilly, Uttar Predesh. (Table No.1 showing the details of patients, i.e., name, age,
gender and date of sample collection)

Age
Sample Type of Date of
S.no. Name of patient in Sex
number sample collection
years
1. S-1 Gazala Parveen 25 F Urine 4/3/2024
2. S-2 Mohd. Haneef 28 M Urine 4/3/2024
3. S-3 Manisha 21 F Urine 4/3/2024
4. S-4 Seema 55 F Urine 4/3/2024
5. S-5 Sunita 36 F Urine 4/3/2024
6. S-6 Naseem 55 F Urine 6/3/2024
7. S-7 Amaan 22 M Urine 6/3/2024
8. S-8 Poonam 43 F Urine 7/3/2024
9. S-9 Ravi 55 M Urine 7/3/2024
10. S-10 Junaid 22 M Urine 9/3/2024
11. S-11 Mubeen 55 M Urine 9/3/2024
12. S-12 Bhagwati 65 F Urine 9/3/2024
13. S-13 Anita 58 F Urine 9/3/2024
14. S-14 Padma 19 F Urine 9/3/2024
15. S-15 Premlata 34 F Urine 9/3/2024
16. S-16 Kanak 42 F Urine 9/3/2024
17. S-17 Raghuveer 56 M Urine 12/3/2024
18. S-18 Nargees 22 F Urine 14/3/2024
19. S-19 Usha 30 F Urine 14/3/2024
20. S-20 Pooja 25 F Urine 14/3/2024
21. S-21 Gultaj 27 F Urine 14/3/2024
22. S-22 Sanjana Maurya 27 F Urine 14/3/2024
23. S-23 Sharda 60 F Urine 16/3/2024
24. S-24 Nehal 32 M Urine 19/3/2024
25. S-25 Sunita 45 F Urine 19/3/2024
26. S-26 Ashu 27 F Urine 19/3/2024
27. S-27 Brahma Devi 80 F Urine 19/3/2024
28. S-28 Omkar Singh 49 M Urine 19/3/2024
29. S-29 Ashna 22 F Urine 20/3/2024
30. S-30 Tara Devi 68 F Urine 20/3/2024
31. S-31 Shalu 35 F Urine 20/3/2024
32. S-32 Neha 28 F Urine 20/3/2024
33. S-33 Asif 72 M Urine 20/3/2024
34. S-34 Sukhdev Tiwari 76 M Urine 21/3/2024
35. S-35 Sheetal Khanna 30 F Urine 21/3/2024
36. S-36 Puru Arora 25 M Urine 21/3/2024
37. S-37 Apoorv Sharma 27 M Urine 21/3/2024
38. S-38 Deepali 58 F Urine 21/3/2024
39. S-39 Prakash 61 M Urine 21/3/2024
40. S-40 Trisha 22 F Urine 21/3/2024
41. S-41 Deeksha 29 F Urine 21/3/2024
42. S-42 Meena 40 F Urine 3/4/2024
43. S-43 Somvati 78 F Urine 3/4/2024
44. S-44 Raajo Devi 81 F Urine 3/4/2024
45. S-45 Baby 46 F Urine 3/4/2024
46. S-46 Prince 23 M Urine 6/4/2024
47. S-47 Dhananjay Singh 45 M Urine 6/4/2024
48. S-48 Anisha 27 F Urine 6/4/2024
49. S-49 Yamuna Devi 65 F Urine 6/4/2024
50. S-50 Neelam 52 F Urine 6/4/2024
51. S-51 Himadri 25 F Urine 8/4/2024
52. S-52 Alok 34 M Urine 8/4/2024
53. S-53 Shagun Sharma 24 F Urine 8/4/2024
54. S-54 Shail Devi 79 F Urine 8/4/2024
55. S-55 Tolaram 67 M Urine 8/4/2024
56. S-56 Pratyush 27 M Urine 8/4/2024
57. S-57 Vaishali 19 F Urine 8/4/2024
58. S-58 Ashutosh 35 M Urine 8/4/2024
59. S-59 Maithili 30 F Urine 8/4/2024
60. S-60 Beena 57 F Urine 8/4/2024
61. S-61 Shashi 30 F Urine 12/4/2024
62. S-62 Sachin 28 M Urine 12/4/2024
63. S-63 Anil 42 M Urine 12/4/2024
64. S-64 Kulveer 45 M Urine 12/4/2024
65. S-65 Nanadram 46 M Urine 12/4/2024
66. S-66 Kanti Devi 50 F Urine 12/4/2024
67. S-67 Satish 35 M Urine 12/4/2024
68. S-68 Shakuntala 43 F Urine 12/4/2024
69. S-69 Sahana 30 F Urine 12/4/2024
70. S-70 Zahida 35 F Urine 12/4/2024
71. S-71 Kasana 27 F Urine 12/4/2024
72. S-72 Qamar 45 F Urine 12/4/2024
73. S-73 Mahendra Patel 57 M Urine 12/4/2024
74. S-74 Maya 22 F Urine 12/4/2024
75. S-75 Rida 26 F Urine 12/4/2024
76. S-76 Shobha 45 F Urine 12/4/2024
77. S-77 Jahan Afroz 36 M Urine 12/4/2024
78. S-78 Ashamaya 40 F Urine 12/4/2024
79. S-79 Poonam 49 F Urine 12/4/2024
80. S-80 Gauri 18 F Urine 12/4/2024
81. S-81 Shaziya 30 F Urine 12/4/2024
82. S-82 Sameer 30 M Urine 12/4/2024
83. S-83 Soni 22 F Urine 12/4/2024
84. S-84 Preet Singh 26 F Urine 12/4/2024
85. S-85 Muradi 60 M Urine 12/4/2024
86. S-86 Raunak 20 M Urine 16/4/2024
87. S-87 Savita 45 F Urine 16/4/2024
88. S-88 Rachna 26 F Urine 16/4/2024
89. S-89 Ramkishan 70 M Urine 16/4/2024
90. S-90 Kamini 55 F Urine 16/4/2024
91. S-91 Yogita 54 F Urine 16/4/2024
92. S-92 Ashwini 48 F Urine 16/4/2024
93. S-93 sudeep 60 M Urine 16/4/2024
94. S-94 Varalika 25 F Urine 16/4/2024
95. S-95 Shilpi Maurya 28 F Urine 12/7/2024
Table No. 1- Sample Collection

Isolation media.
Different types of enrichment and selective media were used for the isolation and identification
of bacteria.

(i) MacConkey Lactose Agar:


Composition (Gm/L)
Peptone (Pancreatic digest of Gelatin) 17.00
Proteose peptone (meat and Casein) 3.000
Lactose monohydrate 10.00
Sodium chloride 5.00
Bile salts 1.50
Neutral red 0.030
Crystal violet 0.001
Agar 13.500
Final pH (at 25℃) 7.1 +/- 0.2

49.53 grams of MacConkey agar was suspended in 1000 ml distilled water and boil for 1 minute
with constant stirring. Sterilize by autoclaving at 15 lbs pressure (121°C at 15 minutes. Avoid
overheating. Cool to 45-50°C. Mix well before pouring into sterile petri plates.

(ii) Nutrient Media Agar


Composition (Gm/L)
Peptone 3.00
Beef extract 15.00
Agar 1.00
Distilled water 5.00
pH (at 25℃) 7.4 +/- 0.2

28.0 grams of nutrient agar media was suspended in 1000 ml purified/distilled water. Heat it
boiling to dissolve the medium completely. Sterilize by autoclaving at 10 lbs pressure (115°C) for
30 minutes or alternatively at 15 lbs pressure (121°C) for 15 minutes.

(iii) Cystine-Lactose-Electrolyte-Deficient (Cled) Agar


Composition (Gm/L)
Peptone 4.000
Tryptone 4.000
HM Peptone B 3.000
Lactose 10.00
L-Cystine 0.128
Bromothymol Blue 0.020
Agar 15.000
Final pH (at 25℃) 7.3 +/- 0.2
Suspend 36.15 grams in 1000 mL of distilled water/ purified water. Heat to boiling to dissolve the
medium completely. Sterilize by autoclaving at 15 lbs pressure (1210 C) for 15 minutes. Cool to
45-500C mix well and pour into sterile petri plates.

Isolation of bacteria
Bacteria were isolated from the urine samples by inoculating them on the culture media
MacConkey using streaking method at incubated at 37℃ for 24-48 hrs. After culturing all the
samples were again sub-cultured on fresh petri plates to isolate the pure colonies.

Identification of bacteria
Cultural observation:-
Colour, size, and colony morphology are observed from the incubated plates.

Microscopic Examination of Urine Specimen:-


Slides were prepared from each different colonies observed on the plates and gram staining was
performed. The results such as the gram positive or gram negative, shape of the bacteria is
observed from the examinations.

Microbiological Analysis Of Urine Specimen:-


The uropathogens were identified by swabbing/streaking the urine specimens on various selective
and differential media such as Hi-Chrome UTI agar, Baird ParkerAgar, Blood Agar, Cetrimide
agar, MacConkey agar based on their colour morphology after the incubationperiod.

Biochemical Characterization of bacteria:-


IMViC Test
E.coli and other bacteria can be confirmed biochemically by the use of a traditional method
called IMViC tests. This is a set of four tests that are used to differentiating bacteria especially
the members of the family Enterobacteriaceae. IMViC is an acronym for four different
biochemical test; each alphabet except ‘i’(added for ease of pronunciation) is for rhyming
purpose. The lower case "i" is merely for "in" as the citrate test requires coliform.

“I” = Indole Test


“M” = Methyl Red (MR) Test
“V” = Voges – Proskauer (VP) Test
“C” = Citrate Utilization Test

Indole Test
Bacteria utilize various amino acids as their food. Tryptophane, one of the amino acids,
may be utilized by certain bacteria producing indole as its by product. Indole can be detected in
the medium by the colourimetric test on addition of suitable reagents.
To carry out this test, it is essential to have tryptophane in the medium. This is nor- mally
present in most of the proteins. Casein digest invariably contains tryptophane and can be used
more satisfactorily than other proteins.

MATERIALS
Peptone water cultures of bacteria, peptone water tubes, ether and Ehrlich's para-di-methyl-
amino-benzaldehyde reagents.
Composition (Gm/L)
p-dimethylamine benzaldehyde 50 gm
Amyl alcohol 750 ml
Hydrochloric acid 250 ml

PROCEDURE
1. Inoculate two tubes of peptone water with bacterial culture.
2. Incubate both inoculated tubes at 37°C for 48 hours.
3. Add 1 ml. of ether to 5 ml of bacterial culture. Shake well and allow to stand till ether collects
on the surface of the medium.
4. Run down 1 ml. of Ehrlich's reagent by the side of the tube and watch for the development of
deep red colour.

INTERPRETATION
The presence of indole is indicated by the development of a deep red colour in the layer of the
reagent.

Methyl Red Test


This test depends upon the ability of certain organisms to produce acidity upto a pH of 4.2
or less by the breakdown of glucose present in the medium. The colour of the medium will be
distinctly red after the addition of methyl red indicator. Some organisms may produce sufficient
acidity to lower the pH to 4.2 in the beginning but subsequently metabolize the acids and give rise
to various neutral products (e.g. ethyl alcohol, acetylmethylcarbinol, diacetyl etc.). The duration
of incubation period of the culture is therefore very important to conduct the test.

MATERIALS
Peptone water cultures bacteria; glucose phosphate peptone water tubes (5 ml.) methyl red
indicator.
Composition (Gm/L)
Methyl red 0.200
Ethyl alcohol 60.00
Distilled water 40.00

PROCEDURE
1. Inoculate two tubes of glucose phosphate peptone water with bacterial culture.
2. Incubate inoculated tubes at 37°C for 48 hours.
3. Add 5 drops of methyl red indicator in each culture tube and also in one uninoculated control
tube for comparing the colours.

INTERPRETATION
Red colour indicates positive reaction and yellow colour negative reaction. Intermediate shades
may be considered as doubtful.

Voges-Proskauer (VP) Test


This test depends upon the ability of the organisms to produce acids from glucose and
subsequently to convert them to a neutral product acetyl methyl carbinol. The addition of alkali
followed by vigorous shaking of the tube, acetyl methyl carbinol is oxidised to diacetyl. Diacetyl
reacts with the guinidine group of arginine which is present in the medium. This reaction produces
a pink colour.
All V. P. positive organisms produce acids first and hence may given a positive M. R. test
in the early stages of the incubation period. If the incubation period is short, the V. P. test may or
may not be given. On longer incubation period, MR test may become negative and VP test
positive.

MATERIALS
Peptone water cultures of bacteria; glucose phosphate peptone water tubes,5% alcoholic solution
of alphanaphthol and 40% aqueous solution of potassium hydroxide.

Voges-Proskauer Reagent A: Barritt's reagent A

Composition (Gm/L)
Alpha-Naphthol(1-Naphthol) 50 gm
Absolute ethanol 1000 ml

Voges-Proskauer Reagent B: Barritt's reagent B

Composition (Gm/L)
Potassium Hydroxide 400 gm
Deionized Water 1000 ml

PROCEDURE
1. Inoculate two tube of glucose phosphate peptone water with bacterial cultures.
2. Incubate the inoculated tubes at 37°C for 48 to 72 hours.
3. Add 0.6 ml. of alpha-naphthol and 0.2 ml. of pot. hydroxide solutions to 1 ml. of culture. Shake
thoroughly and wait for few minutes for the colour to develop at or near the surface.

INTERPRETATION
Positive reaction is indicated by the development of a bright cherry red colour after 5 to 15 minutes
or longer. Negative reaction will show reagent colour.

Citrate Utilization Test


Citrate agar is used to test an organism's ability to utilize citrate as a source of energy. The
process takes place via the enzymes is called citrase. The citrase enzyme hydrolyses the citrate to
form oxaloacetic acid and acetic acid. The test is also called Simmon's citrate test asit utilizes
Simmon's citrate agar that contains citrate as the sole carbon source and inorganic ammonium
salts (NH4H2PO4) as the sole source of nitrogen. The test organism is cultured in a medium which
contains sodium citrate and indicator bromothymol blue. Change in colour ofindicator from light
green to blue due to alkaline reaction is indication of citrate utilization by the test organism.

Composition (Gm/L)
Sodium Chloride 5.000
Magnesium sulphate 0.200
Ammonium dihydrogen phosphate 1.000
Dipotassium phosphate 1.000
Sodium citrate 2.000
Bromothymol blue 0.080
Agar 15.00
Distilled water 1.000
Final pH (at 25℃) 6.9 +/- 0.2

1. Prepare Simmon’s citrate agar medium in test tubes, taking 5 ml medium by autoclaving at 15
lbs for 15 minutes.
2. Tilt the test tube containing melted citrate medium to prepare distinct slant and butt.
3. Streak the slant back and forth with a light inoculum picked from the centre of a well- isolated
colony.
4. Incubate aerobically at 35 to 37 degree C for up to 4-7 days.
5. Observe a colour change from green to blue along the slant.

INTERPRETATION
Positive reaction shows growth with colour change from green to intense blue along the
slant while negative reaction shows no growth and no colour change and slant remains green.

Catalase Test
This is an oxidative enzyme capable of decomposing hydrogen peroxide to water and
oxygen. It is present in most of the aerobic bacteria and removes which is otherwise toxic to the
cells.

MATERIALS

Cultures of bacteria; nutrient agar slants, H₂O₂ solution (10 volumes).


PROCEDURE
1. Inoculate nutrient agar slants with bacterial cultures.
2. Incubate inoculated slants at 37°C for 48 hours.
3. Pour 0.5 ml. H₂O, over the surface of each bacterial culture and observe the effervescence.

INTERPRETATION
If catalase is present, bubbles of oxy- gen are released from the surface of the growth.

Oxidase Test
There are a number of oxidases which can utilize oxygen directly. They contain copper or iron as
the part of their molecule. The ability to produce this enzyme is characteristic of only a few
bacteria.

MATERIALS
Cultures of bacteria; blood agar plates; freshly prepared oxidase reagent (one percent para-amino-
dimethyl aniline-monochloride).
PROCEDURE
1. Inoculate blood agar plates with bacterial cultures.
2. Incubate inoculated plates at 37°C for 48 hours.
3. Cover a portion of each culture with the oxidase reagent and after a minute or two pour off the
excess of the reagent. Watch for the change in colour of the colonies.
INTERPRETATION
Within a few minutes, the colonies change to pink, red and gradually turns to black if oxidase is
present.

Urease Test
Some organisms produce the enzyme urease which hydrolyses urea contained in the
medium. Decomposition of urea is useful for differentiation of certain closely related bacteria.
Urea is easily destroyed by heat and hence sterilized by means of filtration.

MATERIALS
Cultures of bacteria and urea medium slants,
Composition (Gm/L)
Dextrose 1.0
Peptic digest of animal tissue 1.5
Sodium chloride 5.0
Monopotassium phosphate 2.0
Phenol red 0.012
Agar 15.0
Distilled water 1.00
Final pH (at 25°C) 6.8 ±0.2

PROCEDURE
1. Inoculate urea medium slants with bacterial cultures.
2. Incubate the slants at 37°C for 24 to 48 hours.
INTERPRETATION
Urea hydrolysis increases alkalinity which is indicated by the change of the colour of the medium
to pink in presence of phenol red indicator.

Triple Sugar Iron (TSI) Test


Most bacteria have the ability to ferment carbohydrates, particularly sugars. Among them,
each bacterium can ferment only some of the sugars, while it cannot ferment the others. Thus, the
sugars, which a bacterium can ferment, and the sugars, which it cannot is the characteristic of the
bacteria and thus an important criterion for its identification. The Triple Sugar Iron (TSI) agar is
a culture medium named for its ability to test a microorganism's ability to ferment sugars and to
produce hydrogen sulfide.

Composition (Gm/L)
Pancreatic Digest of Casein 15.0
Lactose 10.0
Sucrose 10.0
Sodium chloride 5.0
Peptic digest of animal tissue 5.0
Yeast Extract 3.0
Beef Extract 3.0
Dextrose 1.0
Ferric Ammonium Citrate 0.5
Sodium Thiosulfate 0.3
Phenol red 0.024
Agar 12.0
Distilled water 1.00
Final pH (at 25°C) 7.3 +/- 0.2

1. With a straight inoculation needle, touch the top of a well-isolated colony.


2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube and
then streaking the surface of the agar slant.
3. Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 18 to 24 hours.
4. Examine the reaction of medium.

INTERPRETATION:
An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose fermentation only.
An acid/acid (yellow slant/yellow butt) reaction: It indicates the fermentation of dextrose,
lactose and/or sucrose.
An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate fermentation
results.
Blackening of the medium: Occurs in the presence of H2S.
Gas production: Bubbles or cracks in the agar indicate the production of gas (formation of CO2
and 02).

Carbohydrate Fermentation Test


The carbohydrate fermentation test is a laboratory technique used to identify bacteria
based on their ability to ferment specific carbohydrates. It is used to determine the ability of
bacteria to ferment various carbohydrates, such as sugars, and produce acid or gas.
Composition (Gm/L)
Proteose peptone 10.000
HM Peptone B 1.000
Sodium Chloride 5.000
Phenol Red 0.018
Final pH (at 25°C) 7.4+/- 0.2

PROCEDURE:
1. Prepare a series of tubes containing different carbohydrates (e.g., glucose, lactose, sucrose, etc.)
and a pH indicator (e.g., phenol red).
2. Inoculate each tube with the test bacterium.
3. Incubate the tubes at a suitable temperature (usually 37°C) for 24-48 hours.
4. Observe the tubes for changes in color (indicating acid production) or the presence of gas
bubbles.
INTERPRETATION:
- Acid production (pH change): indicates fermentation of the carbohydrate.
- Gas production (bubbles): indicates fermentation of the carbohydrate.
- No change: indicates the bacterium cannot ferment the carbohydrate.

Extraction Of Plants For Antibacterial Activity:

Sample Collection and Preparation

Dried roots of Salvadora persica (miswak) were bought from local sellers at sailani market,
Bareilly. The chewing sticks samples were washed under running tap water to remove dirt. The
samples were air-dried for 2 days to remove moisture and reduce water activity which inhibits
microbial growth and biochemical reactions. The dried samples were well grinded into a fine
powder with a mixer grinder. The powder was stored in air tight aseptic containers for subsequent
use.
Methods used to prepare Extracts
For the preparation of plant extracts various type of methods are used from which the extract is
obtained. Out of which we used two types of methods.

Maceration: In this extraction process, a container is filled with coarsely powdered


drug material, such as leaves, stem bark, or root bark. The menstruum is poured on top of the
medication material until it is fully covered. After that, the container is closed and left for a
minimum of three days. (Ingle et.al., 2017). Periodically shaking the bottle after adding the
contents will guarantee full extraction. Following the extraction process, the micelle and marc
are separated by decantation or filtration as shown in (figure 3). The micelle is then
evaporatively separated from themenstruum in an oven or on top of a water bath (Azwanida,
2015).

Fig: 3. Maceration process


Soxhlet extraction: Another name for this procedure is continuous hot extraction. The
glass-based device is known as a Soxhlet extractor. Round-bottom flask, extraction chamber,
siphon tube, and condenser at the top make up this apparatus. A plant material that has been
dried, ground, and finely powdered is put inside a porous bag (thimble) that is made of sturdy
filter paper or clean fabric and fastened shut. A bottom flask is filled with the extraction solvent,
and then the extraction chamber is filled with the thimble. After being heated from the bottom
flask, the solvent evaporates, flows through the condenser, flows down to the extraction
chamber, and contacts the drug to extract it. As a result, the extracted plant material and solvent
return to the plant when the solvent level in the extraction chamber reaches the top of the siphon.
Until the medication is fully extracted—that is, when a solvent exiting the extraction chamber
leaves no residue behind—the entire procedure is repeated repeatedly as shown in (figure 4).
This approachworks well for plant material that is partially soluble in the selected solvent as well
as for plantmaterial that is insoluble; thermolabile plant material should not be used with this
procedure (Majekodunmi, 2015).

Fig: 4. Soxhlet Extraction


Preparation of Extracts
The methanolic and ethanolic extracts of miswak sticks were prepared using the soxhlet
extraction and maceration method respectively with slight modifications. The dried stem was
crushed into fine powder.
Ethanolic Extract Using Maceration
About 50g of the powder were separately soaked in 200ml of 70% ethanol in a 250ml reagent
bottle and stoppered. This was allowed to stand for 3-4 days in a dark well tighten bottle with
frequent shaking to permit full extraction of the active principles in the powdered chewing
sticks. The fluids were then filtered using Whatmann No.1 filter paper. The plant macerate was
evaporated using Soxhlet extractor to remove solvent. It was then kept in the fridge at 4ºC.

Figure 7
Maceration process

Figure 8
Evaporation of solvent from extract

Methanolic Extract Using Soxhlet Extraction


A fine powder was extracted in methanol (v/v), using a soxhlet extractor. Dried extracts were
weighed, dissolved in methanol and utilized for the antibacterial activity test.
Figure 9 (b)

Figure 9 (c) Soxhlet extraction

Antibiotic Susceptibility of Plant Extracts:


The failure of conventional antibiotics to completely eradicate bacterial infections in recent
years due to heightened bacterial resistance is a strong motivator to investigate novel medicines
and conquer bacterial resistance. Plant extracts provide more advantages than chemically
derived medications when used therapeutically to control resistant bacteria. Because these
medications have less adverse effects than those derived from chemical sources, they have been
selected as an alternate strategy.
The antibiotic susceptibility was done by the Disk diffusion method on Muller Hinton
Agar to determine the susceptibility and the resistance of the bacteria in order to assist in
selecting the treatment options for the patients.

Procedure:
1. Firstly, Muller-Hinton Agar plates are prepared in well sterilized conditions.
2. Pick a single colony from the culture plate with the help of sterilised cotton swab and
prepare a lawn culture on the MHA plates.
3. Divide the inoculated plates in five compartments i.e.,one compartment for the control
(streptomycin disk) and other four for the plant extract (M.E.M, M.E.N, M.E.B, E.E.M).
4. Impregnate the sterile disks by dipping the sterile disks into the plant extracts.
5. By the help of the Kirby-Bauer’s Disk Diffusion method the impregnated disks are
placed in the culture plate inoculated with the bacteria using forceps.
6. Incubate the plates for 24 hrs at 37℃.
7. After incubation measure the diameter of the zone formed with the help of zone
inhibition scale.
Chapter-IV
Results

Sample Collection:
The present study was undertaken on 103 patients of urinary tract infection of either sex (both
male and female) from hospitals and diagnostic laboratories in Bareilly. Samples were collected
in sterile urine containers, and transferred to Department of Microbiology, MJPRU, Bareilly,
Uttar Pradesh.

Our statistical study makes it abundantly evident that women are more likely than males
to get a urinary tract infection, and that the 20–30 age range is the one with the highest infection
risk. Out of 103 samples we found 11 sterile samples and 92 infected samples from which 8 are
unidentified and 84 known samples are infected by bacteria and candida.

Table no. 2: showing the number and percentage of males and females of different age groups
infected with urinary tract infection.

Table no. 2: Distribution of patients according to age

Age in years No. Of Male Percentage Female Percentage


patients
10-20 04 01 25% 03 75%
21-30 36 08 22% 28 77%
31-40 11 05 45% 06 54%
41-50 16 05 31% 11 68%
51-60 14 06 42% 08 57%
61-70 06 03 50% 03 50%
71-80 05 02 40% 03 60%
81-90 01 00 00% 01 100%
40
35
30
25
20
15
10
5
0
Oct-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90

No. Of patients Male Female

Graph no.1: Frequency of patients according to their age.

Table 3: Distribution of UTI pathogens with sex of the patients

S.
Organism isolated Total Male Percentage Female Percentage
no.
1. E.coli 19 5 26% 14 74%
2. Klebsiella 27 7 26% 20 74%
3. Staphylococcus 13 4 31% 9 69%
4. Proteus 2 0 0% 2 100%
5. Pseudomonas 8 3 37% 5 63%
6. Salmonella 3 2 66% 1 34%
7. Candida 11 1 9% 10 91%
30

25

20

15

10

Total Male Female

Graph no.2: frequency of pathogens with age of the patients

Table 4: On the Basis of Microbes isolated from the collected sample:

Sample Age in Type of Date of


S. no. Sex Interpretation
number years sample collection
1. S-1 25 F Urine 4/3/2024 E.coli
2. S-2 28 M Urine 4/3/2024 E.coli
3. S-3 21 F Urine 4/3/2024 -
Klebsiella
4. S-4 55 F Urine 4/3/2024
pneumoniae
Klebsiella
5. S-5 36 F Urine 4/3/2024
pneumoniae
6. S-6 55 F Urine 6/3/2024 -
Staphylococcus
7. S-7 22 M Urine 6/3/2024
aureus
Staphylococcus
8. S-8 43 F Urine 7/3/2024
aureus
9. S-9 55 M Urine 7/3/2024 -
10. S-10 22 M Urine 9/3/2024 -
11. S-11 55 M Urine 9/3/2024 -
12. S-12 65 F Urine 9/3/2024 E.coli
13. S-13 58 F Urine 9/3/2024 E.coli
14. S-14 19 F Urine 9/3/2024 E.coli
Staphylococcus
15. S-15 34 F Urine 9/3/2024
aureus
16. S-16 42 F Urine 9/3/2024 E.coli
17. S-17 56 M Urine 12/3/2024 E.coli
Klebsiella
18. S-18 22 F Urine 14/3/2024
pneumoniae
Klebsiella
19. S-19 30 F Urine 14/3/2024
pneumoniae
Klebsiella
20. S-20 25 F Urine 14/3/2024
pneumoniae
21. S-21 27 F Urine 14/3/2024 Proteus mirabilis
Staphylococcus
22. S-22 27 F Urine 14/3/2024
aureus
Klebsiella
23. S-23 60 F Urine 16/3/2024
pneumoniae
24. S-24 32 M Urine 19/3/2024 E.coli
Staphylococcus
25. S-25 45 F Urine 19/3/2024
aureus
Klebsiella
26. S-26 27 F Urine 19/3/2024
pneumoniae
27. S-27 80 F Urine 19/3/2024 -
Klebsiella
28. S-28 49 M Urine 19/3/2024
pneumoniae
Klebsiella
29. S-29 22 F Urine 20/3/2024
pneumoniae
30. S-30 68 F Urine 20/3/2024 Proteus mirabilis
31. S-31 35 F Urine 20/3/2024 Salmonella
32. S-32 28 F Urine 20/3/2024 E.coli
Staphylococcus
33. S-33 72 M Urine 20/3/2024
aureus
34. S-34 76 M Urine 21/3/2024 -
35. S-35 30 F Urine 21/3/2024 E.coli
36. S-36 25 M Urine 21/3/2024 Salmonella sp.
Klebsiella
37. S-37 27 M Urine 21/3/2024
pneumoniae
Staphylococcus
38. S-38 58 F Urine 21/3/2024
saprophyticus
Staphylococcus
39. S-39 61 M Urine 21/3/2024
aureus
Staphylococcus
40. S-40 22 F Urine 21/3/2024
aureus
Klebsiella
41. S-41 29 F Urine 21/3/2024
pneumoniae
42. S-42 40 F Urine 3/4/2024 -
43. S-43 78 F Urine 3/4/2024 -
44. S-44 81 F Urine 3/4/2024 Candida albicans
45. S-45 46 F Urine 3/4/2024 -
46. S-46 23 M Urine 6/4/2024 -
Klebsiella
47. S-47 45 M Urine 6/4/2024
pneumoniae
48. S-48 27 F Urine 6/4/2024 E.coli
Klebsiella
49. S-49 65 F Urine 6/4/2024
pneumoniae
Klebsiella
50. S-50 52 F Urine 6/4/2024
pneumoniae
Klebsiella
51. S-51 25 F Urine 8/4/2024
pneumoniae
52. S-52 34 M Urine 8/4/2024 Not identify
Staphylococcus
53. S-53 24 F Urine 8/4/2024
aureus
Klebsiella
54. S-54 79 F Urine 8/4/2024
pneumoniae
Klebsiella
55. S-55 67 M Urine 8/4/2024
pneumoniae
Klebsiella
56. S-56 27 M Urine 8/4/2024
pneumoniae
57. S-57 19 F Urine 8/4/2024 Not identify
58. S-58 35 M Urine 8/4/2024 Not identify
59. S-59 30 F Urine 8/4/2024 Not identify
60. S-60 57 F Urine 8/4/2024 Not identify
Klebsiella
61. S-61 30 F Urine 12/4/2024
pneumoniae
Klebsiella
62. S-62 28 M Urine 12/4/2024
pneumoniae
Staphylococcus
63. S-63 42 M Urine 12/4/2024
aureus
64. S-64 45 M Urine 12/4/2024 Salmonella sp.
65. S-65 46 M Urine 12/4/2024 E.coli
66. S-66 50 F Urine 12/4/2024 Not identify
Staphylococcus
67. S-67 35 M Urine 12/4/2024
aureus
68. S-68 43 F Urine 12/4/2024 E.coli
69. S-69 30 F Urine 12/4/2024 E.coli
70. S-70 35 F Urine 12/4/2024 E.coli
Staphylococcus
71. S-71 27 F Urine 12/4/2024
aureus
Klebsiella
72. S-72 45 F Urine 12/4/2024
pneumoniae
73. S-73 57 M Urine 12/4/2024 Not identify
Klebsiella
74. S-74 22 F Urine 12/4/2024
pneumoniae
Pseudomonas
75. S-75 26 F Urine 12/4/2024
aeruginosa
Pseudomonas
76. S-76 45 F Urine 12/4/2024
aeruginosa
Klebsiella
77. S-77 36 M Urine 12/4/2024
pneumoniae
Klebsiella
78. S-78 40 F Urine 12/4/2024
pneumoniae
79. S-79 49 F Urine 12/4/2024 E.coli
Staphylococcus
80. S-80 18 F Urine 12/4/2024
aureus
81. S-81 30 F Urine 12/4/2024 E.coli
82. S-82 30 M Urine 12/4/2024 E.coli
Klebsiella
83. S-83 22 F Urine 12/4/2024
pneumoniae
84. S-84 26 F Urine 12/4/2024 E.coli
Pseudomonas
85. S-85 60 M Urine 12/4/2024
aeruginosa
Pseudomonas
86. S-86 20 M Urine 16/4/2024
aeruginosa
Klebsiella
87. S-87 45 F Urine 16/4/2024
pneumoniae
Klebsiella
88. S-88 26 F Urine 16/4/2024
pneumoniae
Klebsiella
89. S-89 70 M Urine 16/4/2024
pneumoniae
90. S-90 55 F Urine 16/4/2024 Not identify
Candida
91. S-91 54 F Urine 16/4/2024
parapsilosis
Pseudomonas
92. S-92 48 F Urine 16/4/2024
aeruginosa
Pseudomonas
93. S-93 60 M Urine 16/4/2024
aeruginosa
Pseudomonas
94. S-94 25 F Urine 16/4/2024
aeruginosa
95. S-95 45 F Urine 18/4/2024 Candida albicans
96. S-96 38 F Urine 18/4/2024 Candida albicans
97. S-97 32 F Urine 18/4/2024 Candida albicans
Candida
98. S-98 45 F Urine 18/4/2024
tropicalis
99. S-99 30 F Urine 18/4/2024 Candida krusei
Candida
100. S-100 44 F Urine 18/4/2024
parapsilosis
Candida
101. S-101 52 F Urine 23/04/2024
parapsilosis
102. S-102 32 F Urine 23/04/2024 Candida glabrata
Candida
103. S-103 48 M Urine 23/04/2024
tropicalis

Percentage of Microbes
9%
21%
12%

8%

2%
3%
30%

15%

E.coli Klebsiella Staphylococcus Salmonella


Proteus Pseudomonas Candida Not Identify

Figure 10:- Representing the types of Microorganisms isolated from the urine samples:

Isolation and identification:


(Cultural techniques)

Figure11:- Lactose fermenting bacteria on MacConkey appears pink

Figure no.11: Lactose fermenting colonies on MacConkey appears pink

Figure12:- Non-Lactose Fermenting Bacteria on MacConkey appears pale yellow


E.coli Staphylococcus Klebsiella Pseudomonas Proteus Salmonella
On
MacCo
nkey
Agar

Colony Pink to dark Pale pink to red Mucoid, large, Circular, Smooth,pal Colorless,
Charact pink colonies, lactose pink to pale pink mucoid and e,or convex, 2-
ers colonies, fermenting. colonies colorless colourless 3mm with
dry and colonies with a serrate
donut swarming margin
shaped,
colonies
surrounded
by a pink
area of bile
salts.

Figure no. 13: Different colony characters on MacConkey agar; Pink to dark pink colonies,
dry and donut shaped, surrounded by a pink area of bile salts indicates E.coli, Pale pink to red
colonies, lactose fermenting indicates Staphylococcus, Mucoid, large, pink to pale pink
colonies indicates Klebsiella, Circular, mucoid and colorless colonies indicates Pseudomonas.
Smooth,pale,or colourless with swarming colonies indicates Proteus and Colorless, convex, 2-
3mm with a serrate margin indicates Salmonella.
E.coli Candida Klebsiella Pseudomonas
On Cled
Agar

Colony Yellow lactose- Pale yellow White to bluish mucoid Circular, mucoid
Characters positive colonies colonies colonies and colorless
colonies

Figure 14: Different bacterial colony characters on Cled Agar; Yellow lactose fermenting
colonies are of E.coli, pale yellow colonies are of Candida, white to bluish mucoid colonies are
of Klebsiella and circular, mucoid and colorless colonies are of pseudomonas.

(Gram Staining: Microscopy)

Figure no. 15(a) Figure no. 15(b)


Figure no. 15(c) Figure no. 15(d)

Figure15:- Gram’s Staining showing negative and positive microorganisms; fig no. 15(a)
Gram negative thick rods. (b) Gram negative thin rods. (c) Gram positive cocci. (d)
Gram positive oval shaped (candida).

Table no. 6: Biochemical Tests Interpretation

Biochemical Test Positive Negative


Indole 21 62
Methyl Red Test 71 12
Voges-Proskeur Test 14 69
Citrate Utilization Test 53 29
Catalase Test 73 10

Table no. 7: Results Of IMViC and Catalase Test


Sample
S.no. Indole MR VP Citrate Catalase Interpretation
number
1. S-1 +ve +ve -ve -ve +ve E.coli
2. S-2 +ve +ve -ve -ve +ve E.coli
3. S-3 - - - - - -
Klebsiella
4. S-4 -ve +ve +ve +ve +ve
pneumoniae
Klebsiella
5. S-5 -ve +ve +ve +ve -ve
pneumoniae
6. S-6 - - - - - -
Staphylococcus
7. S-7 -ve -ve -ve +ve +ve
aureus
Staphylococcus
8. S-8 -ve +ve -ve -ve +ve
aureus
9. S-9 - - - - - -
10. S-10 - - - - - -
11. S-11 - - - - - -
12. S-12 +ve +ve -ve -ve +ve E.coli
13. S-13 +ve +ve -ve -ve +ve E.coli
14. S-14 +ve +ve -ve -ve +ve E.coli
Staphylococcus
15. S-15 -ve +ve +ve +ve +ve
aureus
16. S-16 +ve +ve -ve -ve +ve E.coli
17. S-17 +ve +ve -ve -ve +ve E.coli
Klebsiella
18. S-18 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
19. S-19 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
20. S-20 -ve +ve -ve +ve +ve
pneumoniae
21. S-21 -ve +ve -ve +ve +ve Proteus mirabilis
Staphylococcus
22. S-22 -ve +ve +ve +ve +ve
aureus
Klebsiella
23. S-23 -ve +ve -ve +ve +ve
pneumoniae
24. S-24 +ve +ve -ve -ve +ve E.coli
Staphylococcus
25. S-25 -ve +ve +ve +ve +ve
aureus
Klebsiella
26. S-26 -ve +ve -ve +ve +ve
pneumoniae
27. S-27 - - - - - -
Klebsiella
28. S-28 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
29. S-29 -ve +ve -ve +ve +ve
pneumoniae
30. S-30 -ve +ve -ve +ve +ve Proteus mirabilis
31. S-31 -ve +ve -ve +ve +ve Salmonella
32. S-32 +ve +ve -ve -ve +ve E.coli
Staphylococcus
33. S-33 -ve +ve +ve +ve +ve
aureus
34. S-34 - - - - - -
35. S-35 +ve +ve -ve -ve +ve E.coli
36. S-36 -ve +ve -ve +ve +ve Salmonella sp.
Klebsiella
37. S-37 -ve +ve -ve +ve +ve
pneumoniae
Staphylococcus
38. S-38 -ve -ve -ve -ve +ve
saprophyticus
Staphylococcus
39. S-39 -ve +ve -ve +ve +ve
aureus
Staphylococcus
40. S-40 -ve +ve -ve +ve +ve
aureus
Klebsiella
41. S-41 -ve +ve -ve +ve +ve
pneumoniae
42. S-42 - - - - - -
43. S-43 - - - - - -
44. S-44 -ve +ve -ve -ve +ve Candida albicans
45. S-45 - - - - - -
46. S-46 - - - - - -
Klebsiella
47. S-47 -ve +ve -ve +ve +ve
pneumoniae
48. S-48 +ve +ve -ve +ve +ve E.coli
Klebsiella
49. S-49 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
50. S-50 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
51. S-51 -ve +ve -ve +ve +ve
pneumoniae
52. S-52 - - - - - Not identify
Staphylococcus
53. S-53 -ve +ve +ve +ve +ve
aureus
Klebsiella
54. S-54 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
55. S-55 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
56. S-56 -ve +ve -ve +ve +ve
pneumoniae
57. S-57 +ve -ve -ve -ve -ve Not identify
58. S-58 -ve +ve +ve -ve +ve Not identify
59. S-59 +ve +ve -ve +ve +ve Not identify
60. S-60 +ve +ve -ve +ve -ve Not identify
Klebsiella
61. S-61 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
62. S-62 -ve +ve -ve -ve -ve
pneumoniae
Staphylococcus
63. S-63 -ve +ve +ve +ve +ve
aureus
64. S-64 -ve +ve -ve +ve +ve Salmonella sp.
65. S-65 +ve +ve -ve +ve +ve E.coli
66. S-66 -ve -ve +ve -ve +ve Not identify
Staphylococcus
67. S-67 -ve +ve +ve +ve +ve
aureus
68. S-68 +ve +ve -ve -ve +ve E.coli
69. S-69 +ve +ve -ve -ve +ve E.coli
70. S-70 +ve +ve -ve -ve +ve E.coli
Staphylococcus
71. S-71 -ve +ve +ve +ve +ve
aureus
Klebsiella
72. S-72 +ve +ve -ve +ve +ve
pneumoniae
73. S-73 -ve +ve -ve -ve -ve Not identify
Klebsiella
74. S-74 -ve +ve -ve +ve +ve
pneumoniae
Pseudomonas
75. S-75 -ve -ve -ve +ve +ve
aeruginosa
Pseudomonas
76. S-76 -ve -ve -ve +ve +ve
aeruginosa
Klebsiella
77. S-77 -ve +ve -ve +ve +ve
pneumoniae
Klebsiella
78. S-78 -ve +ve -ve -ve -ve
pneumoniae
79. S-79 +ve +ve -ve -ve +ve E.coli
Staphylococcus
80. S-80 -ve +ve +ve +ve +ve
aureus
81. S-81 +ve +ve -ve -ve +ve E.coli
82. S-82 +ve +ve -ve -ve -ve E.coli
Klebsiella
83. S-83 -ve +ve -ve +ve +ve
pneumoniae
84. S-84 +ve +ve -ve -ve +ve E.coli
Pseudomonas
85. S-85 -ve -ve -ve +ve +ve
aeruginosa
Pseudomonas
86. S-86 -ve -ve -ve +ve +ve
aeruginosa
Klebsiella
87. S-87 -ve +ve -ve -ve +ve
pneumoniae
Klebsiella
88. S-88 -ve +ve -ve +ve -ve
pneumoniae
Klebsiella
89. S-89 -ve +ve -ve +ve +ve
pneumoniae
90. S-90 -ve -ve -ve -ve +ve Not identify
Candida
91. S-91 -ve +ve +ve +ve -ve
parapsilosis
Pseudomonas
92. S-92 -ve -ve -ve +ve +ve
aeruginosa
Pseudomonas
93. S-93 -ve -ve -ve +ve +ve
aeruginosa
Pseudomonas
94. S-94 -ve -ve -ve +ve +ve
aeruginosa
95. S-95 -ve +ve -ve -ve +ve Candida albicans
96. S-96 -ve +ve -ve -ve +ve Candida albicans
97. S-97 -ve +ve -ve -ve +ve Candida albicans
98. S-98 +ve +ve +ve +ve +ve Candida tropicalis
99. S-99 -ve -ve -ve +ve -ve Candida krusei
100. S-100 Candida
-ve +ve +ve +ve +ve
parapsilosis
101. S-101 Candida
-ve +ve +ve +ve +ve
parapsilosis
102. S-102 -ve +ve -ve -ve -ve Candida glabrata
103. S-103 +ve +ve +ve +ve +ve Candida tropicalis
+ve = positive
-ve = negative

Indole Test:

Figure no. 17: Indole test- red ring shows positive indole test result and yellow ring shows
negative test result.
Methyl-Red Test:

Figure no.18: Methyl-red test - red ring shows positive result and yellow ring shows negative
result foe MR test.

Voges-Proskeur Test:

Figure no. 19: Voges-Prosker Test :- red ring shows positive results and yellow rings shows
negative results for VP test.

Citrate Utilization Test:


Figure no. 20: Citrarte Utilization Test:- blue colour of the slant shows positive result and
green colour of the slant shows negative results for citrate utilization test.

Catalase Test:

Figure no. 21: Catalase test:- bubbles are formed on adding H2O2 indicates positive result
and no bubbles formation indicates negative results for catalase test

Table no. 8: Results of Urease Test


S.no. Sample number Urease Interpretation
1. S-1 -ve E.coli
2. S-2 -ve E.coli
3. S-3 - -
4. S-4 +ve Klebsiella pneumoniae
5. S-5 +ve Klebsiella pneumoniae
6. S-6 - -
7. S-7 +ve Staphylococcus aureus
8. S-8 +ve Staphylococcus aureus
9. S-9 - -
10. S-10 - -
11. S-11 - -
12. S-12 -ve E.coli
13. S-13 -ve E.coli
14. S-14 -ve E.coli
15. S-15 +ve Staphylococcus aureus
16. S-16 -ve E.coli
17. S-17 -ve E.coli
18. S-18 +ve Klebsiella pneumoniae
19. S-19 +ve Klebsiella pneumoniae
20. S-20 +ve Klebsiella pneumoniae
21. S-21 +ve Proteus mirabilis
22. S-22 +ve Staphylococcus aureus
23. S-23 +ve Klebsiella pneumoniae
24. S-24 -ve E.coli
25. S-25 +ve Staphylococcus aureus
26. S-26 +ve Klebsiella pneumoniae
27. S-27 - -
28. S-28 +ve Klebsiella pneumoniae
29. S-29 +ve Klebsiella pneumoniae
30. S-30 +ve Proteus mirabilis
31. S-31 -ve Salmonella
32. S-32 -ve E.coli
33. S-33 +ve Staphylococcus aureus
34. S-34 - -
35. S-35 -ve E.coli
36. S-36 -ve Salmonella sp.
37. S-37 +ve Klebsiella pneumoniae
38. S-38 +ve Staphylococcus saprophyticus
39. S-39 +ve Staphylococcus aureus
40. S-40 +ve Staphylococcus aureus
41. S-41 +ve Klebsiella pneumoniae
42. S-42 - -
43. S-43 - -
44. S-44 +ve Candida albicans
45. S-45 - -
46. S-46 - -
47. S-47 +ve Klebsiella pneumoniae
48. S-48 -ve E.coli
49. S-49 +ve Klebsiella pneumoniae
50. S-50 +ve Klebsiella pneumoniae
51. S-51 +ve Klebsiella pneumoniae
52. S-52 - Not identify
53. S-53 +ve Staphylococcus aureus
54. S-54 +ve Klebsiella pneumoniae
55. S-55 +ve Klebsiella pneumoniae
56. S-56 +ve Klebsiella pneumoniae
57. S-57 -ve Not identify
58. S-58 +ve Not identify
59. S-59 +ve Not identify
60. S-60 -ve Not identify
61. S-61 +ve Klebsiella pneumoniae
62. S-62 +ve Klebsiella pneumoniae
63. S-63 +ve Staphylococcus aureus
64. S-64 -ve Salmonella sp.
65. S-65 -ve E.coli
66. S-66 +ve Not identify
67. S-67 +ve Staphylococcus aureus
68. S-68 -ve E.coli
69. S-69 -ve E.coli
70. S-70 -ve E.coli
71. S-71 +ve Staphylococcus aureus
72. S-72 +ve Klebsiella pneumoniae
73. S-73 -ve Not identify
74. S-74 +ve Klebsiella pneumoniae
75. S-75 +ve Pseudomonas aeruginosa
76. S-76 +ve Pseudomonas aeruginosa
77. S-77 +ve Klebsiella pneumoniae
78. S-78 +ve Klebsiella pneumoniae
79. S-79 -ve E.coli
80. S-80 +ve Staphylococcus aureus
81. S-81 -ve E.coli
82. S-82 -ve E.coli
83. S-83 +ve Klebsiella pneumoniae
84. S-84 -ve E.coli
85. S-85 +ve Pseudomonas aeruginosa
86. S-86 +ve Pseudomonas aeruginosa
87. S-87 +ve Klebsiella pneumoniae
88. S-88 +ve Klebsiella pneumoniae
89. S-89 +ve Klebsiella pneumoniae
90. S-90 +ve Not identify
91. S-91 -ve Candida parapsilosis
92. S-92 +ve Pseudomonas aeruginosa
93. S-93 +ve Pseudomonas aeruginosa
94. S-94 +ve Pseudomonas aeruginosa
95. S-95 -ve Candida albicans
96. S-96 -ve Candida albicans
97. S-97 -ve Candida albicans
98. S-98 +ve Candida tropicalis
99. S-99 +ve Candida krusei
100. S-100 -ve Candida parapsilosis
101. S-101 -ve Candida parapsilosis
102. S-102 +ve Candida glabrata
103. S-103 -ve Candida tropicalis

+ve = positive; -ve = negative


Urease Test:

Figure no. 22: Result of urease test showing pink colour for positive result indicates
microorganism hydrolyses urea and yellow colour for negative results for urease test.

Table no. 9: Results of Triple Sugar Iron Test


Sample Gas H 2S
S.no. Slant Butt Interpretation
number production production
1. S-1 yellow yellow +ve -ve E.coli
2. S-2 yellow Yellow +ve -ve E.coli
3. S-3 - - - - -
Klebsiella
4. S-4 red yellow +ve -ve
pneumoniae
Klebsiella
5. S-5 red yellow +ve -ve
pneumoniae
6. S-6 - - - - -
Staphylococcus
7. S-7 red red -ve -ve
aureus
Staphylococcus
8. S-8 red red -ve -ve
aureus
9. S-9 - - - - -
10. S-10 - - - - -
11. S-11 - - - - -
12. S-12 yellow yellow +ve -ve E.coli
13. S-13 yellow yellow +ve -ve E.coli
14. S-14 yellow yellow +ve -ve E.coli
Staphylococcus
15. S-15 yellow red +ve -ve
aureus
16. S-16 yellow yellow +ve -ve E.coli
17. S-17 red red +ve +ve E.coli
Klebsiella
18. S-18 yellow yellow +ve -ve
pneumoniae
Klebsiella
19. S-19 yellow yellow +ve -ve
pneumoniae
Klebsiella
20. S-20 red red -ve -ve
pneumoniae
21. S-21 red yellow -ve -ve Proteus mirabilis
Staphylococcus
22. S-22 yellow red +ve -
aureus
Klebsiella
23. S-23 yellow yellow +ve -ve
pneumoniae
24. S-24 yellow yellow -ve -ve E.coli
Staphylococcus
25. S-25 red red -ve -ve
aureus
Klebsiella
26. S-26 yellow dark +ve +ve
pneumoniae
27. S-27 - - - - -
Klebsiella
28. S-28 yellow yellow +ve -ve
pneumoniae
Klebsiella
29. S-29 red red -ve -ve
pneumoniae
30. S-30 yellow dark +ve +ve Proteus mirabilis
31. S-31 red yellow -ve +ve Salmonella
32. S-32 yellow yellow +ve -ve E.coli
Staphylococcus
33. S-33 red yellow +ve -ve
aureus
34. S-34 - - - - -
35. S-35 yellow yellow +ve -ve E.coli
36. S-36 red red -ve -ve Salmonella sp.
Klebsiella
37. S-37 yellow dark +ve +ve
pneumoniae
Staphylococcus
38. S-38 red red -ve -ve
saprophyticus
Staphylococcus
39. S-39
aureus
Staphylococcus
40. S-40 yellow yellow -ve -ve
aureus
Klebsiella
41. S-41 red yellow +ve +ve
pneumoniae
42. S-42 - - - - -
43. S-43 - - - - -
44. S-44 red yellow -ve -ve Candida albicans
45. S-45 - - - - -
46. S-46 - - - - -
Klebsiella
47. S-47 yellow red +ve -ve
pneumoniae
48. S-48 yellow yellow +ve -ve E.coli
Klebsiella
49. S-49 red yellow +ve -ve
pneumoniae
Klebsiella
50. S-50 red yellow +ve -ve
pneumoniae
Klebsiella
51. S-51 yellow yellow +ve -ve
pneumoniae
52. S-52 - - - - Not identify
Staphylococcus
53. S-53 red yellow +ve -ve
aureus
Klebsiella
54. S-54 yellow yellow +ve -ve
pneumoniae
Klebsiella
55. S-55 yellow yellow -ve -ve
pneumoniae
Klebsiella
56. S-56 yellow red +ve -ve
pneumoniae
57. S-57 red red -ve -ve Not identify
58. S-58 yellow yellow +ve -ve Not identify
59. S-59 red red -ve -ve Not identify
60. S-60 yellow yellow -ve -ve Not identify
Klebsiella
61. S-61 red yellow -ve -ve
pneumoniae
Klebsiella
62. S-62 yellow yellow -ve -ve
pneumoniae
Staphylococcus
63. S-63 red yellow -ve -ve
aureus
64. S-64 red red -ve -ve Salmonella sp.
65. S-65 yellow red +ve -ve E.coli
66. S-66 red red -ve -ve Not identify
Staphylococcus
67. S-67 red yellow -ve -ve
aureus
68. S-68 yellow yellow +ve -ve E.coli
69. S-69 yellow yellow +ve -ve E.coli
70. S-70 yellow yellow +ve -ve E.coli
Staphylococcus
71. S-71 red yellow -ve -ve
aureus
Klebsiella
72. S-72 yellow yellow -ve -ve
pneumoniae
73. S-73 red red -ve -ve Not identify
Klebsiella
74. S-74 yellow yellow -ve -ve
pneumoniae
Pseudomonas
75. S-75 red red -ve -ve
aeruginosa
Pseudomonas
76. S-76 red red -ve -ve
aeruginosa
Klebsiella
77. S-77 red yellow -ve -ve
pneumoniae
Klebsiella
78. S-78 yellow yellow -ve -ve
pneumoniae
79. S-79 red red -ve -ve E.coli
Staphylococcus
80. S-80 yellow yellow +ve -ve
aureus
81. S-81 yellow yellow +ve -ve E.coli
82. S-82 yellow yellow -ve +ve E.coli
Klebsiella
83. S-83 yellow yellow +ve -ve
pneumoniae
84. S-84 yellow yellow +ve -ve E.coli
Pseudomonas
85. S-85 red red -ve -ve
aeruginosa
Pseudomonas
86. S-86 red red -ve -ve
aeruginosa
Klebsiella
87. S-87 red red -ve -ve
pneumoniae
Klebsiella
88. S-88 red yellow +ve -ve
pneumoniae
Klebsiella
89. S-89 red red -ve -ve
pneumoniae
90. S-90 yellow yellow +ve -ve Not identify
91. S-91 red yellow -ve -ve Candida parapsilosis
Pseudomonas
92. S-92 red red -ve -ve
aeruginosa
Pseudomonas
93. S-93 red red -ve -ve
aeruginosa
Pseudomonas
94. S-94 red red -ve -ve
aeruginosa
95. S-95 red yellow -ve -ve Candida albicans
96. S-96 red yellow -ve -ve Candida albicans
97. S-97 red yellow -ve -ve Candida albicans
98. S-98 red yellow -ve -ve Candida tropicalis
99. S-99 red yellow -ve -ve Candida krusei
100. S-100 red yellow -ve -ve Candida parapsilosis
101. S-101 red yellow -ve -ve Candida parapsilosis
102. S-102 red yellow -ve -ve Candida glabrata
103. S-103 red yellow -ve -ve Candida tropicalis

+ve = positive; -ve = negative


Triple Sugar Iron Test:

Figure no. 23.Results of TSI test showing colour changes from red to yellow indicating
acid production, gas production (bubbles) and H2S Production as blackening of the bottom.

Table no. 10 Results of Carbohydrate Fermentation Test


Bacteria
Sugars
Klebsiella E.coli Staphylococcus Pseudomonas Proteus Salmonella
Maltose +ve +ve +ve -ve -ve -ve
Dextrose +ve +ve +ve +ve +ve +ve
Inulin +ve -ve +ve -ve -ve -ve
Fructose +ve +ve +ve +ve -ve -ve
Sucrose +ve -ve +ve -ve -ve -ve
Inositol +ve -ve -ve -ve -ve -ve
Lactose +ve +ve +ve -ve -ve -ve
Trehalose +ve +ve +ve -ve +ve +ve
Adonitol +ve -ve -ve -ve -ve -ve
Galactose +ve +ve +ve -ve -ve -ve
Mannose +ve +ve +ve -ve -ve +ve

+ve = positive; -ve = negative


Carbohydrate Fermentation Test:-

Figure no. 24. Results of carbohydrate fermentation test showing colour changes from red to
yellow indicating particular sugar is fermented

Molecular Characterization of Yeast Isolates


The yeast strains isolated during this study morphological analysis cannot be sufficient
to identify the yeast isolates accurately. Therefore, molecular analysis is further expected to
characterise them accurately. Molecular identification of 02 yeast isolates was carried out based
on conserved Ribosomal ITS regions. The ITS Region of each yeast isolates between the small
nuclear 18srDNA and large nuclear 28s rDNA, including 5.8s rDNA was amplified using a pair
of universal primers, ITSI and ITS4. Figure shows the PCR products of the 02 isolates with
expected sizes of 550-650 bp on the 1% agarose gel electrophorersis. The identities of the
fungal isolates were confirmed by sequencing the PCR amplified product of ITS regions.
BLAST Search in NCBI Gene Bank database using the sequence of ITS regions revealed that
S44, S91 isolates had 100, 100%, homology with, Candida albicans, Candida parapsilosis
respectively. (Table- 6). The ITS sequences of the 02 Isolates S44, S91(s), were deposited to
NCBI Gene Bank under the assertion numbers KU60020.115 and IFM 61745 respectively.
(Table no.11 )

Table No.-11 Identification of yeast strains isolated from infected urine samples based on
morphological criteria as well as genetic analysis of the internal transcribed spacer (ITS)
region. The closest relatives to yeast strains according to BLAST search are presented. In
case the BLAST search yielded a cultured but undesignated strain as the closest relative, the
closest cultured and the designated relative are given additionally.

Strain Morphological Sequence Next related Assertion No. Similarity(%)


identification lenth(bp) yeast isolates
strain(BLAST)
S44 Candida 547bp Candida KU60020.115 100%
albicans albicans
S91 Candida 547bp Candida IFM 61745 100%
parapsilosis parapsilosis

Sequencing of PCR products and BLAST and Phylogeny analysis


The PCR products were sequenced bi-directionally. The sequences generated in the study
are shown in figure 26-27. The generated sequence data was aligned and analyzed using
BLAST (Basic Local Alignment Search Tool) to identify similar fungal sequences. The
percent identity of yeast isolates with similar organisms is shown in Table 11.

S-44: The sequencing for ITS region of S-44 sample generated a sequence of 547 bp.
BLAST analysis with other sequences in GenBank found the S-44 sequence to have
100% identity with Candida albicans isolate KU60020.115. The phylogentic tree for
the sample is shown in figure 26.

S-91: The sequencing for ITS region of S-91 sample generated a sequence of 547 bp.
BLAST analysis with other sequences in GenBank found the S-91 sequence to have
100% identity with Candida parapsilosis IFM 61745. The phylogentic tree for the
sample is shown in figure 27.
~1000bp

~500bp

Figure No 28. Gel Electrophoresis of the PCR Product of 02 yeast isolates performed by
ITS1 (F) and ITS4 (R) primers and showing ˜500bp amplification in well 11 &12.

Table no. 12: Antimicrobial Activity of Plant Extracts Using Disk Diffusion
Methods

Sample number Methanolic Ethanolic Methanolic Methanolic Control


extract of extract of extract of extract of (Streptomycin)
miswak miswak nirgundi banyan
S-01 (E.coli) 10mm - - - 10mm
S-02 (E.coli) 11mm - - - 10mm
S-12 (E.coli) 10mm - - - 11mm
S-14 (E.coli) 11mm - - - -
S-24 (E.coli) 10mm - - - 15mm
S-32 (E.coli) 10mm 11mm - - 11mm
S-35 (E.coli) 10mm - - - 11mm
S-65 (E.coli) 10mm - - - 10mm
S-69 (E.coli) 12mm - - - 10mm
S-70 (E.coli) 10mm - - - 10mm
S-79 (E.coli) 10mm - - - 10mm
S-82 (E.coli) 10mm 10mm - - 10mm
S-54 (Klebsiella) 11mm - - - 12mm
S-72 (Klebsiella) 11mm - - - 12mm
S-21 (Proteus) 10mm - - - 14mm
S-75 18mm - - - -
(Pseudomonas)
S-76 10mm - - - -
(Pseudomonas)
S-94 10mm - - - 15mm
(Pseudomonas)
S-07 11mm - - - -
(Staphylococcus)
S-08 10mm - - - -
(Staphylococcus)
S40 11mm - - 10mm -
(Staphylococcus)
S-44 18mm - 10mm 13mm 36mm
(Staphylococcus)

Figure no. 29: Sterile disc is impregnated on bacterial lawn culture with plant extract and
zones are formed after incubation for 24 hours at 370C

Table no. 13: Antimicrobial Resistance Pattern For Bacterial Isolates


Results
Microorganisms Antimicrobial Resistance Pattern
GEN CEC AZM CD AMP C
Staphylococcus S R R R R S
E.coli R R R R R R
Klebsiella R R R R R R
Proteus S S S R R R
Pseudomonas S S R R R R
Salmonella S S R R R R
GEN: Gentamicin; CEC: Clavulanic acid; AZM: Azithromycin; CD: Clindamycin; AMP:
Ampicillin; C: Chloramphenicol

Figure no. 30(a) Figure no. 30(b)

Figure no. 30: Antimicrobial resistance pattern showing susceptibility and resistance pattern
on MHA zones are formed around antibiotic discs after incubation at temperature 370C for 24
hours . Figure no. 30(a): Antibiotic disc are placed on bacterial lawn cultures after incubation
zones are formed i.e., susceptible. Figure no. 30(b) very less zones or no zones are formed
after incubation i.e., resistant.

Table no.14:- Antimicrobial Resistance Pattern For Yeast Isolates Results:


S.no. Sample Antimicrobial Resistance Pattern
no. NS CC KT AP IT FLC
1. S-44 R R R R R R
2. S-91 S R S S S R
3. S-95 S S R S R R
4. S-96 R R R R R R
5. S-97 S S S S R R
6. S-98 S R R R R R
7. S-99 S R R R R R
8. S-100 S R R R R R
9. S-101 R S S S R R
10. S-102 R R R R R R
11. S-103 S S S S R R

NS: Nystatin; CC: Clotrimazole; KT: Ketoconazole; AP: Amphotericin B; IT: Itraconazole;
FLC: Fluconazole
Table no. 14: Antimicrobial Activity of Plant Extracts Using Well Diffusion
Method Using Different Concentration Results

Antibacterial Activity Of Plant Extract On Resistant Bacteria

Bacterial Methanolic extract of Methanolic extract of Methanolic extract of Ethanolic extract of

Samples Nirgundi Banyan Miswak Miswak

20% 25% 50% 20% 25% 50% 20% 25% 50% 20% 25% 50%

Proteus 14mm 15mm 15mm 17mm 20mm 21mm - - - - - -


Staphylococcus - - - - - - 17mm 18mm 19mm 15mm 16mm 14mm
Pseudomonas - - - 17mm 18mm 16mm - - - - - -
Klebsiella - - - - - - - - - - - -
E.coli - - - - - 10mm 12mm 16mm 17mm - - -
Salmonella - - - - 13mm 13mm - - - - 14mm 16mm

Antimicrobial Activity Of Plant Extracts Using Well Diffusion Method


Using Different Concentration

Figure no.31 (a) Figure no.31 (b)


no.33333331: (a)
Figure no. 31: Antibacterial activity of plant extract of miswak, banyan and nirgundi with 20%, 25%
and 50% concentrations using well diffusion method. Fig no. 31(a) promising results are shown by
different concentrations banyan extract and very less zones are formed by different extract of nirgundi
on Proteus mirabilis. Fig no.31(b) zones are formed by different concentrations of methanolic and
ethanolic extract of miswak on Staphylococcus aureu
Chapter-V
Discussion
One of the biggest threats to international health in the twenty-first century is bacterial infection
(Rios et.al., 1998). Antibiotic-resistant bacteria are a serious health concern, therefore finding
new medicines with innovative modes of action is essential to combating this issue (Wang
et.al., 2003). Plants have historically offered hope for new therapeutic molecules because plant-
based herbal remedies have significantly improved human health and wellbeing (Iwu et.al.,
1999). For therapeutic treatments, the application of plant extracts with established antibacterial
qualities can be very important.
Antibiotic susceptibility test results revealed that almost all of the isolates were resistant
to the majority of the antibiotics used in the study. There is a claim that the frequency and types
of antibiotic-resistant strains in humans are directly correlated with the type of antibiotic that
was used. Transmissible elements and plasmids are an easy way for bacteria to spread
antimicrobial agent resistance.
Insufficient development of novel therapeutic agents and inadequate medication
policies have led to the daily expansion of drug-resistant bacteria, despite the tremendous
advancements in modern science. In order to address this issue, professionals from a variety of
disciplines, including medicine, pharmacy, microbiology, molecular biology, and
phytochemistry, are collaborating to identify natural resources that may contain strong
antibacterial activity candidates that could be effective in combating pathogenic
microorganisms.
The findings of our study unveiled that some commonly available, medicinal plant in
India are highly effective against the pathogenic bacteria isolated from the patients suffering
from Urinary Tract Infection (UTI), which is one of the most common disease not only in India
but also in other developing and developed countries. We studied the effect of three different
plant extracts we studies: Salvadora persica (miswak), Vitex negundo (nirgundi) and Ficus
benghalensis (banyan). While the ethanolic extract of miswak and methanolic extract of
nirgundi showed minimum antibacterial activity, the methanolic activity of the miswak and
banyan showed the highest antibacterial activity against the UTI-causing bacteria. This
suggests that the specific bioactive compounds present in the methanol extracts have strong
efficacy against the UTI-causing bacteria.
Some bacterial isolates obtained from UTI such as Proteus mirabilis, Staphylococcus
aureus, E.coli, Pseudomonas and Salmonella are found to be multi drug resistant (MDR) againt
antibiotic, i.e., Gentamicin, Chloramphenicol, Ciprofloxacin, Ampicillin, Clindamycin,
Azithromycin. The plant extract were used to determined the antibacterial activity againt these
MDR bacteria.
Proteus mirabilis was susceptible to methanolic extract of nirgundi and banyan
exhibiting a zone of inhibition ranging from 13-15mm and 17-21mm respectively with
concentration of 20%, 25%, and 50%.
Staphylococcus aureus was susceptible to methanolic and ethanolic extract of miswak
exhibiting a zone of inhibition ranging from 17-19mm and 14-16mm respectively with
concentration of 20%, 25%, and 50%.
Pseudomonas aeruginosa was susceptible to methanolic extract of banyan exhibiting
a zone of inhibition ranging from 16-18mm with concentration of 20%, 25%, and 50%.
Escherichia coli was susceptible to methanolic extract of miswak exhibiting a zone of
inhibition ranging from 12-17mm with concentration of 20%, 25%, and 50%.
Salmonella was susceptible to methanolic extract of banyan and ethanolic extract of
miswak exhibiting a zone of inhibition ranging 13mm and 14-16mm respectively with
concentration of 25% and 50%.
From the study we conclude that most of the plants showed antimicrobial activity against one
or more UTI isolates. The broad spectrum activity was observed in miswak and banyan. The
antibacterial potential of these plants againt UTI causing pathogens have been reported earlier
and needs extensive investigation to understand their antibacterial principles which may allow
the scientific community to recommend their use as accessible alternative to synthetic
antibiotics.
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