Antibacterial Activity of Herbal Plants Popularly Used in India (1)
Antibacterial Activity of Herbal Plants Popularly Used in India (1)
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.
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).
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).
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).
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.
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.
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 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,
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).
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).
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.
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.
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.
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.
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.
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.
MATERIALS
Peptone water cultures of bacteria; glucose phosphate peptone water tubes,5% alcoholic solution
of alphanaphthol and 40% aqueous solution of potassium hydroxide.
Composition (Gm/L)
Alpha-Naphthol(1-Naphthol) 50 gm
Absolute ethanol 1000 ml
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.
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
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.
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
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).
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.
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.
Figure 7
Maceration process
Figure 8
Evaporation of solvent from extract
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.
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
Percentage of Microbes
9%
21%
12%
8%
2%
3%
30%
15%
Figure 10:- Representing the types of Microorganisms isolated from the urine samples:
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.
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).
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.
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
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.
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.
Figure no. 24. Results of carbohydrate fermentation test showing colour changes from red to
yellow indicating particular sugar is fermented
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.
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
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
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.
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
20% 25% 50% 20% 25% 50% 20% 25% 50% 20% 25% 50%
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