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Extraction, phytochemical screening and activity evaluation of Eichhornia crassipes leaves extracts against gram-negative and gram-positive antibiotic-resistance isolates

Authors:
  • Al-shatrah University
  • Independent Researcher

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It has been observed that the leaves of Eichhornia crassipes have a significant capacity to absorb water from rivers, leading to water loss and potentially blocking the flow of rivers. However, previous studies provided that the Eichhornia crassipes has biological important in the environment. The objective of this study is to isolate and identify pathogenic bacterial species from patients at AS-Shatrah Hospital, to examine the antibacterial properties of Eichhornia crassipes leaf extracts against specific pathogenic bacterial species, including Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeroginosa, and Escherichia coli. Furthermore, the study seeks to investigate the metabolic activity of these pathogenic isolates in utilizing 13 different sugars and 10 amino acids. The plant leaves were collected and subjected to phytochemical examination in order to identify their individual components. The antibacterial properties of the ethanol extracts were evaluated using the Plate diffusion technique. The results suggest that ethanol extracts have a substantial effect on both bacterial and multidrug-resistant (MDR) bacterial strains, particularly when compared to the antibiotic used as a control. The study's findings indicated that the ethanol extract obtained from water hyacinth exhibits significant antibacterial and antioxidant activities. The observed effects can be attributed to the existence of numerous biologically active constituents, such as flavonoids, alkaloids, and phenols. Consequently, this research suggests that water hyacinth, despite being considered a causing harm aquatic herb, holds potential as an alternative source of compounds for therapeutic applications. It is employed as a means of controlling bacterial infections and minimizing the impact of infectious diseases, and reduces the consumption of antibiotics as well.
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*Corresponding author: haidersa@shu.edu.iq https://doi.org/10.36547/ae.2024.6.2.28-36
Abstract
It has been observed that the leaves of Eichhornia crassipes have a significant capacity to absorb water from
rivers, leading to water loss and potentially blocking the flow of rivers. However, previous studies provided
that the Eichhornia crassipes has biological important in the environment. The objective of this study is to
isolate and identify pathogenic bacterial species from patients at AS-Shatrah Hospital, to examine the
antibacterial properties of Eichhornia crassipes leaf extracts against specific pathogenic bacterial species,
including Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeroginosa, and Escherichia coli.
Furthermore, the study seeks to investigate the metabolic activity of these pathogenic isolates in utilizing
13 different sugars and 10 amino acids. The plant leaves were collected and subjected to phytochemical
examination in order to identify their individual components. The antibacterial properties of the ethanol
extracts were evaluated using the Plate diffusion technique. The results suggest that ethanol extracts have
a substantial effect on both bacterial and multidrug-resistant (MDR) bacterial strains, particularly when
compared to the antibiotic used as a control. The study's findings indicated that the ethanol extract obtained
from water hyacinth exhibits significant antibacterial and antioxidant activities. The observed effects can
be attributed to the existence of numerous biologically active constituents, such as flavonoids, alkaloids,
and phenols. Consequently, this research suggests that water hyacinth, despite being considered a causing
harm aquatic herb, holds potential as an alternative source of compounds for therapeutic applications. It is
employed as a means of controlling bacterial infections and minimizing the impact of infectious diseases,
and reduces the consumption of antibiotics as well.
Archives of Ecotoxicology, Vol. 6, No. 2, pp. 28-36, 2024
Extraction, phytochemical screening and activity evaluation of Eichhornia
crassipes leaves extracts against gram-negative and gram-positive antibiotic-
resistance isolates
Haider Sabah Abdulhuseina*, Baidaa Mezher Kadimb
a Department of Pathological Analysis, College of Applied Science, Shatrah University, Iraq
b Department of Microbiology, College of Veterinary Medicine, Shatrah University, Iraq
1. Introduction
Eichhornia crassipes, commonly referred to as water hyacinth, is
an aquatic plant that grows quickly and is distinguished by its
large fibrous root systems and significant biomass. It is abundant
in different tropical and subtropical regions worldwide (Gao
and Bo, 2004). Many plant species have the capacity to act as
valuable reservoirs of important antibacterial properties.
Consequently, the utilization of phytochemicals and plant
extracts, which are acknowledged for their established
antibacterial qualities, assumes an essential function in
treatment strategies (Sanaa, et al., 2012). The examination of
alcoholic plant extract against bacterial species is being done
because to the plant's essential qualities and structural features.
Staphylococcus aureus is a bacterium that is spherical in shape
and has a diameter of around 1μm. It is classified as Gram-
positive. After being incubated for duration of 24 hours, the
microorganism develops clusters resembling grapes. These
colonies display a notable, as well, round, and glossy appearance
on the growth medium. The optimum temperature for its growth
is 37℃, and it flourishes within a pH range of 7.4-7.6.
Staphylococcus aureus is therapeutically significant because it
possesses a variety of virulence factors. These elements
contribute to its capacity to invade host organisms, provoke
pathogenicity, and acquire resistance to drugs. Notably, one such
virulence factor is protein-A, which covalently attaches to the
peptidoglycan layer (Rayan and Ray 2004). Certain strains
possess a capsule consisting of glucosamineuronic acid, which is
lost throughout the process of cultivating. Additionally, these
strains exhibit the capacity to create enzymes such as Protease,
Hyaluronidase, Nuclease, phosphatase, and Fibrinolysin
coagulase (Francois and Screzel 2008) and (Humphreys
1997). Pseudomonas aeruginosa, a Gram-negative rod-shaped
bacterium, is widely distributed in the environment. It is
recognized as a significant pathogen that poses a serious threat
to individuals with compromised immune systems, causing
nosocomial infections, as well as persistent respiratory tract
infections in patients with cystic fibrosis (CF) (Lyczak, 2006).
The capacity to adapt many ecological settings and acquire
resistance to conventional antibiotic treatments has engendered
a new research attention with bacteriophages. Phage mixtures
have been employed by many researchers for the treatment of
Archives of Ecotoxicology
Journal homepage: https://office.scicell.org/index.php/AE
Keywords:
Antibacterial activity,
Eichhornia crassipes,
Flavonoids,
Alkaloids,
Multidrug-resistant
Article info
Received 8 March 2024
Revised 17 June 2024
Accepted 20 July 2024
Published online 3 October 2024
Regular article
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
29
Pseudomonas aeruginosa infections; nonetheless, it is important
to conduct clinical tests in order to assess their effectiveness and
safety (Kutateladze and Adamia 2008). Several antibiotics
have restricted penetration into biofilms (Hoiby et al., 2010).
Hence, it has been observed that bacteria surviving in biofilms
exhibit a remarkable ability to withstand antibiotic doses that
are several orders higher greater than those tolerated by
planktonic bacteria (Ciofu et al., 2002). Over a period of time,
this phenomenon facilitates the emergence of antibiotic-
resistant bacteria and leads to the prevalence of multi-drug-
resistant (MDR) strains (Hirsch and Tam 2010). Streptococcus
pyogenes, sometimes known as group A Streptococcus, is a
frequently noted bacterium. Streptococcus, or GAS, is a type of
Gram-positive cocci that commonly inhabits humans as its
specific host. Streptococcus is a bacterial pathogen that causes a
wide range of infections in humans, varying from mild
conditions like sore throat and impetigo to more severe illnesses
such as necrotizing fasciitis, glomerulonephritis, acute
rheumatic fever, and streptococcal toxic shock syndrome (Luca-
Harari et al., 2009) and (Carapetis et al., 2005). Escherichia
coli, a member of the Enterobacteriaceae family, is a highly
diverse and widespread group. Gram-negative facultative
anaerobic bacilli are bacteria with dimensions ranging from
around 0.3 to 1.0 μm in width and 1.0 to 6.0 μm in length. The
bacteria possess peritrichous flagella, which enables them to
move. The organism demonstrates its highest level of growth
and metabolic activity when the temperature is between 25-
35℃. It is capable of engaging in both respiratory and
fermentative metabolic activities (Public Health England,
2015).
During this study 10 swab samples were obtained from
patients 15 isolates were isolated and diagnosed depending on
biochemical tests and molecular technique, and undergone to
alcoholic extract of Eichhornia leaves. The aim of this study is to
isolate and identify disease-causing bacterial species from
patients at AS-Shatrah Hospital. Additionally, we will investigate
the antibacterial effects of Eichhornia crassipes leaf extracts on
specific disease-causing bacterial species, such as Streptococcus
pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and
Escherichia coli. Moreover, the study aims to examine the
metabolic activity of these disease-causing samples in utilizing a
total of 13 distinct sugars and 10 different amino acids.
2. Material and Methods
2.1 Materials
Eichhornia leaves were collected from the Tigris River/ AL-
Shatrah city/ Iraq. Subsequently, they were subjected to drying
process and afterwards ground until a fine powder. The
resultant powder was then properly stored in a dry container.
The chemicals were acquired from the laboratory involved with
the college. The research was conducted at the organic chemistry
laboratory in the Department of Pharmaceutical Chemistry
Science.
2.2 Preliminary phytochemical screening
The ethanol extract was prepared by soaking 60 grams of
powdered Eichhornia Vulgaris leaves in 300 mL of ethanol for a
period of two weeks. The solution obtained from the extraction
process was subjected to filtration, followed by the evaporation
of ethanol using a rotary evaporator under vacuum conditions at
a temperature of 450°C for duration of one to five hours. The
filtrate was utilized for the purpose of conducting phytochemical
screening, with the objective of confirming the presence of
phytochemicals by carrying out of the tests described by
(Alyaseen, et al., 2018; Abdulridha, et al., 2019).
2.3 Bacteria and media
In according to the procedure described by MacFadden (2000),
specimens were procured, and swabs were carefully obtained
from the site of infection. These swabs were then placed in tubes
containing a medium for preservation, providing that the swabs
remained moist during transportation to the laboratory. A total
of 10 samples were obtained from patients at AS-Shatrah
Hospital, which are represented 2 samples of throat, 3 samples
of Urinary tract, 3 samples of burns, and skin wounds, and 2
samples of respiratory system. Swab specimens were
transferred to the laboratory under aseptic conditions for
analysis. All swab samples were cultured on blood agar, nutrient
agar, and chocolate agar as well. Subsequently, the samples were
divided into two groups based on the gram stain technique,
which is gram positive and gram negative (MacFadden 2000).
2.3.1 Identification of Bacteria
While the samples of Gram negative isolates were cultured on
MacConkey agar and Eosin methylene blue (EMB) (Levine
2018). The plates were incubated at 37℃ for 24 hours. The
Gram-positive specimens were subjected to culture on blood
agar and mannitol saline agar (MSA), in order to investigate their
hemolysis properties and to isolate Staphylococcus, respectively.
2.3.2 Chemical tests:
The pathogenic bacterial isolates were subjected to IMVIC,
catalase, urease, oxidase, coagulase, blood hemolysis, and
mannitol fermentation tests in order to achieve an accurate and
unambiguous diagnosis.
2.3.2.1 Morphological and Microscopic examination
The morphology of colonies was examined using MacConkey
agar, blood agar, EMB and MSA. After incubating at 37˚C for
duration of 24 hours, many characteristics of the colonies were
observed and recorded. These included the color, size, edge
morphology, and the type of hemolysis on blood agar.
Additionally, a single colony of each isolate was fixed onto a clean
slide for the purpose of conducting a gram stain analysis under a
light microscope (Atlas et al., 1995).
The morphology and pigmentation of bacterial colonies were
investigated. This was achieved by observing the shape and
color of colonies on petri plates after they were cultivated and
incubated overnight at a temperature of 37°C (Arahal et al.,
1996; Oren et al., 1997). A sample of bacterial cultures, grown
under optimal conditions, was collected and suspended in
physiological saline solution. A small drop of this bacterial
suspension was placed on a glass slide and gently mixed. The
slide was then passed over a benzene flame and allowed to air
dry for one minutes to facilitate the removal of excess moisture.
Following this, a Gram stain (Crystal violet) was directly applied
onto the bacteria and left for one minute. The slides were
subsequently rinsed with tap water for five seconds to remove
any unbound crystal violet. Next, Gram iodine was added and left
for one minute before being washed with saline solution. A
solution of 95% ethanol was applied for 20 second and washed.
Safranin dye was added as a counter stain. Subsequently, the
sample was cleaned and left to air dry. After that, the slides were
examined by using a light microscope with an immersion lens
and observed the Gram-negative and Gram-positive isolates
(Gram 1884; Sánchez-Porro et al., 2011).
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
30
2.3.2.2 IMVIC
Bacterial growth was injected into test tubes with peptone water
and cultivated at a temperature of 37℃ for one night.
Subsequently, two drops of kovag's reagent were added, and the
appearance of a red or pink color was considered as a positive
result for tryptophanase activity. The methyl red test involves
inoculating glucose phosphate broth with bacterial growth and
incubating it at 37℃ for 48 hours. After incubation, 5 drops of
methyl red are added, and the presence of a red hue indicates a
positive result. The Voges-Proskauer test was conducted using
the same procedure, but with the addition of 0.2 ml of 40% KOH
and alpha-naphthol as the reagent. The positive result was a red
color within one hour. The bacterial inoculum was cultured
overnight in Simmon's citrate agar at a temperature of 37℃.
Subsequently, a positive result was observed as the color of the
agar changed to a deep blue hue (Microbe Online, 2014).
2.3.2.3 Urease, Oxidase, Catalase and Coagulase tests
Inoculation of the urea agar was performed within a test tube,
followed by incubation for duration of 24 hours. The presence of
urease enzyme was inferred by the change in color to pink in the
urea agar test tubes, as reported by (Bilgehan 2014;
Abdulhusein 2023). In the experimental procedure, the
addition of tetra-dimethyl-para-phenylenediamine
dihydrochloride onto the filter paper was followed by the
smearing of bacterial colonies onto the same filter paper.
Subsequently, the results were observed as the appearance of a
blue-violet color within a time frame of 20 seconds (Abdulhusein
2023; Harley, and Prescott 2002). Two drops of hydrogen
peroxide (H2O2) with a concentration of 3% was applied to the
colonies grown on the growth medium. The colonies that
exhibited a positive reaction produced gas bubbles, but the
negative isolates did not demonstrate any bubble formation. The
purpose of this experiment was to differentiate between
Staphylococcus sp. and Streptococcus sp. by utilizing a specialized
test that specifically targets aerobic and microaerophilic
bacteria (Abdulhusein 2023; Harley, and Prescott 2002).. For
coagulase, test tube contains rabbit plasma was inoculated with
bacterial growth and incubated overnight at 37℃, the formation
of clot considered as appositive result; whereas the absence of
clot interpreted as a negative result (Barrow and Feltham,
1993).
2.3.2.4 Blood haemolysis
The isolates were grown on Blood agar, which consisted of a 5%
human blood agar base. Subsequently, the Petri plates were
placed in an incubator set at a temperature of 37℃ for duration
of 24 hours. The isolates were re-identified using the usual
protocols outlined by Cowan (Barrow and Feltham, 1993). The
isolates were classified based on the type of haemolysis
observed around the developed colonies after the incubation
period. The strain that showed a negative catalase test and
exhibited beta haemolysis was identified as Streptococcus
pyogenes.
2.3.2.5 Mannitol Fermentation
Due to its 7.5% sodium chloride (NaCl) content, Mannitol agar
has proven to be a selective medium for particular bacteria, such
as Staphylococcus sp. The isolates cultivated on mannitol salt
agar containing mannose and phenol red showed positive
results for catalase activity and haemolysis after being incubated
for 24 hours at 37℃. Yellow colonies observed on mannitol agar
after the incubation time was considered as indicative of positive
mannitol activity shown by Staphylococcus aureus. The change in
color from red to pink was perceived as a favorable indication of
mannitol action by other Staphylococcus sp. (Santos et al., 2015;
Saab et al., 2018).
2.3.2.6 Utilization of amino acids and carbon sources by
pathogenic bacterial isolates
The metabolic activity of pathogenic bacterial isolates was
evaluated by using 10 amino acids and 13 carbohydrates. The
amino acids including (L-valine, L-methione, L-phenylalanine, L-
serine, L-alanine, Trans-4-hydroxy-L-proline, L-cystine, Glysin,
L-tyrosine, and L-proline) The test tubes incubated at 35℃ for
24 hours after had been inoculated with bacterial isolates
separately (Abdulhusein 2023; Harley, and Prescott 2002).
Using 1% (w/v) (D-(-)-ribose, D-(+)-galactose, Lactose, D-(+)-
trehalose, Maltose, D- Sorbitol, D-(-)-Salicin, D-(+)-melibiose, D-
(+)-xylose, Xylitol, D-(-)-fructose, N-acetylglucose amine, and
Sucrose), the utilization of 13 sugar sources by tested isolates
was examined separately. While production the purple color in
the test tubes accepted as a positive result for utilization amino
acids, the transformation color from red to yellow was
interpreted as a positive result for carbohydrate utilization
(Abdulhusein 2023; Harley, and Prescott 2002).
2.4 McFarland Standard
The McFarland Standards were employed in order to
standardize the estimated quantity of bacteria present in a liquid
suspension. This was achieved by comparing the level of
visibility or turbidity seen in the test suspension with that of the
McFarland Standard (McFarland 1907). The 0.5 McFarland
Standards is the standard employed in clinical microbiology
laboratories for antibiotic susceptibility testing and culture
media performance testing. The turbidity of all suspensions
containing pathogenic bacteria was adjusted to a concentration
of 1×108 CFU/mL, according the directions of McFarland
Standard No 0.5, using sterile saline solution.
2.5 Molecular diagnosis
By using specific extraction and purification kits (QIAamp DNA
and QIAquick PCR Kits, respectively (Qiagen, Germany)),
chromosomal DNA isolated and purified depending on
instructions, then DNA amplified by using PCR technique and
specific forward and reverse primers for Staphylococcus aureus,
[F-(GTAGGTGGCAAGCGTTACC) R-(CGCACATCA GCGTCAG) (Al-
musawi et al., 2014)], for streptococcus pyogenes [F-
(AAGAGAGACTAACGCATG TTAGTAAT) and R-
(ATTTTCCACTCCCACCATCA) (Kulkarni et al., 2016)], for
Pseudomonas aeroginosa [F-(GGGGGATCTTCGGACCTCA) and R-
(TCCTTAGAGTGCC CACCCG) (Spilker et al., 2004)], and for
Escherichia coli [F-(GACCTCGGTTTAGTTCACAGA) and R-
(CACACGCTGACGCTGA CCA) (Mamun et al., 2016)].
2.6 Evaluation the antibacterial activity of Eichhornia
crassipes Leaves Extracts against pathogenic species
Approximately 10 µL of each bacterial suspension, with a
concentration of 1×108 colony-forming units per milliliter, was
applied onto the surface of Muller Hinton Agar (MHA) plates. The
bacterial suspension was spread out using a hokey glass
spreader and was allowed to settle for duration of 2 minutes.
Subsequently, 3 µL of Eichhornia crassipes Leaves Extract was
added to each agar plate. All agar plates were then incubated at
a temperature of 37℃ for the duration of one night. Following
that, the measurement of inhibitory zones was conducted (Rios
et al., 1988).
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
31
3. Results
The result exhibited that from 10 swab specimens 15 isolates
were isolated and depending on the results of Gram staining
reaction these isolates divided into groups. The results of
biochemical tests (Indole, Methyl red, citrate utilization, Vogues
proskauer, blood haemolysis, urease, coagulase, catalase,
oxidase and mannitol fermentation test) proved that all these 15
isolates belonging to four genus, which were including
Staphylococcus (3 isolates), Streptococcus (2 isolates),
Escherichia (7 isolates), and Pseudomonas (3 isolates), (Table 1).
The four genuses were diagnosed as a Gram-positive was
Staphylococcus and Streptococcus and as a Gram-negative were
Pseudomonas and Escherichia, (Table 2).
Table 1 The samples’ codes and bacterial genus, and isolates
number
Isolates’ codes
Bacterial genus
Isolates’ number
SS1*
Escherichia
7
SS2
Pseudomonas
3
SS3
Streptococcus
2
SS4
Staphylococcus
3
*SS: Swab Sample
Table 2 Chemical tests of pathogenic isolates (The final
diagnosis).
Isolates
(SSI3)
(SSI2)
(SSI1)
Bacterial
shape
Cocci/chin
s
Bacillus/ro
d shape
Rod
Gram stain
Positive
Negative
Negative
Catalase
Negative
Positive
Positive
Coagulase
Negative
Negative
Negative
Blood
hemolysis
beta-
hemolysis
β-
Hemolysis
none
Urease
Negative
Positive
Negative
Colonies
shape
circular
low convex
Smooth
Colonies
color
white to
gray
Greenish
blue
Green
metallic
sheen
Motility
Non-Motile
Motile
Motile
Oxidase
Negative
Positive
Negative
Indole
Negative
Negative
Positive
Methyl red
Positive
Negative
Positive
Voges-
Proskauer
Negative
Negative
Negative
Citrate
Negative
Positive
Negative
The result shows that the Staphylococcus sp. (isolate SS4),
Streptococcus sp. (isolate SS3), and Pseudomonas sp. (Isolate
SS2) produce transparent zones around colonies on blood agar,
whereas Escherichia sp. (isolate SS1) does not produce blood
haemolysis, (Table 2). While isolate (SS4) showed Gram-
positive, catalase+, coagulase +, urease+, beta-hemolysis on
blood agar, and oxidase negative. Isolate (SS3) showed Gram-
positive, catalase-, coagulase-, urease-, beta-hemolysis on blood
agar, and oxidase negative. The results of biochemical tests for
isolates SSI and SS2 were Gram stain negative, catalase positive,
and coagulase negative alike. While, isolate SSI exhibited urease
and oxidase negative; isolate SS2 was positive for urease and
oxidase alike. The result of molecular investigation by using
specific 16S rRNA primers found that the isolates isolated from
patients including Staphylococcus aureus, Streptococcus
pyogenes, Pseudomonas aeroginosa, and Escherichia coli.
The results revealed that some of these isolates have ability to
use carbohydrates as a carbon source and change the color of
medium from red to yellow. Ribose, galactose, lactose, maltose,
sorbitol, salicin, D-(+)-melibiose, xylose, fructose, N-
acetylglucose amine, and sucrose were respectively utilized by
50%, 50%, 75%, 75%, 25%, 75%, 75%, 25%, 75%, and 75%.
Although all isolates utilized trehalose 100%, none of the
isolates used xylitol as a carbon source. Furthermore, all amino
acids which used in this study were utilized by bacterial isolates
with different percentages. While, methionine, phenylalanine,
serine, alanine, and proline were utilized by all tested isolates,
50% of our isolates utilized valine, cystine, glysin and Trans-4-
hydroxy-L-proline. Whereas, tyrosine utilized by Staphylococcus,
Pseudomonas, and Escherichia; Streptococcus did not used
tyrosine, (Table 3).
The results of phytochemical screening exhibited that the
chemical structure of Eichhornia consist of alkaloids, flavonoids,
carbohydrate, glycoside, tannin, and Terpenoids by using
Dragendroof reagent and Wanger reagent, Shinoda test, Molish
test, Fehling’s test, FeCl3, and Salkowski reaction, respectively
(Table 4). This study aimed to analyze the presence of
phytochemicals in the leaves of Eichhornia through ethanol
extraction.
The findings of this study indicate that a significant impact of
ethanol extracts on the tested bacteria (Table 5; Figure 2).
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
32
Table 3 Utilization of amino acids and carbohydrates by bacterial isolates
Isolate numbers
Staphylococcus
aureus
Streptococcus
pyogenes
Pseudomonas
aeroginosa
Escherichia
coli
Total
number
Positive
%
L-valine
+
-
-
+
2
50
L-methionine
+
+
+
+
4
100
L-phenylalanine
+
+
+
+
4
100
L-serine
+
+
+
+
4
100
L-alanine
+
+
+
+
4
100
Trans-4-hydroxy-L-proline
-
-
+
+
2
50
L-cystine
-
+
+
-
2
50
Glysin
-
-
+
+
2
50
L-tyrosine
+
-
+
+
3
75
L-proline
+
+
+
+
4
100
D-(-)-ribose
+
-
-
+
2
50
D-(+)-galactose
+
+
+
+
2
50
Lactose
+
+
-
+
3
75
D-(+)-trehalose
+
+
+
+
4
100
Maltose
+
+
-
+
3
75
D- Sorbitol
-
-
-
+
1
25
D-(-)-Salicin
-
+
+
+
3
75
D-(+)-melibiose
+
-
+
+
3
75
D-(+)-xylose
-
-
-
+
1
25
Xylitol
-
-
-
-
0
0
D-(-)-fructose
+
+
-
+
3
75
Sucrose
+
+
-
+
3
75
N-acetylglucose amine
+
-
+
+
3
75
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
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Table 4 Phytochemical screening, chemical structure, chemical test, test result of Thyme Leaves and chemical notes
Table 5 Show the effects of Ethanol extracts of Eichhornia vulgaris leaves on Gr-ve and Gr+ bacteria
No
Isolates
Gram reaction
Ethanol extracts of Eichhornia vulgaris
leaves
Inhibition zone
1
Staphylococcus aureus
Gr+
40 mm
2
Streptococcus pyogenes
Gr+
45 mm
3
Escherichia coli
Gr-
45 mm
4
Pseudomonas aeroginosa
Gr-
50 mm
Figure 1 (A) Blood haemolysis by Staphylococcus aureus blood agar; (B) The colonies of Staphylococcus aureus
Code
No
Chemical structure
Chemical test
Test result of
Thyme leaves
Chemical note
1
Alkaloids
Dragendroff reagent
Wagner reagent
+ve
+ve
formation Orange
Brown ppt
2
Flavonoids
Shinoda test
+ve
formation of pinkish violet color
3
Carbohydrate
Molish test
+ve
Violet color ring formation
4
Glycoside
Fehling’s test
+ve
Blue color formation
5
Tannin
FeCl3
+ve
Bluish black color Formation
6
Saponin
Shaken of the extraction
-ve
No Formation of foam
7
Sterols
Liebermann burchard
-ve
Formation green-blue color
8
Coumarine
Filter paper soaked by diluted NaoH
-ve
No formation of yellowish green color on
filter paper
9
Terpenoids
Salkowski reaction
+ve
Formation of reddish brown color
10
Resins
Ethanol 95% +boiling + 4% hcl
-ve
No formation of turbidity
11
Anthraquinons
Borntrager's test
-ve
No Formation of red rose color
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
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Figure 2 Show the inhibition zone around the extract that affected against bacterial species on Muller Hinton agar
4. Discussion
Earlier study found that the Escherichia coli utilize different
kinds of amino acids in different percentages such as
phenylalanine (Liu et al., 2018), serine (Zhang et al., 2019),
alanine (Xu et al., 2021) and phenylalanine (Satoh et al., 2023).
Our observations differ from findings reported by Baral and
Vaidya in 2011, who found that the aqueous extract exhibited
weak antibacterial activity, compared to other extracts, but
demonstrated better efficacy as an antifungal agent. This
phenomenon can be attributed to the bacteria's lack of prior
exposure to these extracts, resulting in their absence of
resistance. The findings of this study match with a previous
study conducted by Sanaa M. et al. in 2010, as well as with the
results reported by Hiba H. Hamid et al. in 2013 in Iraq. Bacterial
cells and appropriate vectors are employed to introduce
chemicals into the cell, with the aim of inhibiting the activity of
coenzymes and their biologically active compounds
(Harbornne 1984; Mitscher 1992).
These findings elucidate the mechanism underlying the capacity
of multidrug-resistant (MDR) microorganisms to withstand the
effects of antibiotics. The World Health Organization's yearly
reports in 2016 indicate a significant increase in the prevalence
of antimicrobial-resistant bacteria worldwide. Bacteria possess
the capacity to proliferate despite the application of antibiotics
within the leather industry (Berber 2020). In recent decades,
the phenomenon of antibiotic resistance has become
increasingly prevalent as a result of the uncontrolled and
excessive utilization of antimicrobial drugs. Animal products,
poultry, bird feces, and corpses have been identified as
significant sources of multidrug-resistant (MDR) pathogenic
bioagents. Multi-drug resistance (MDR) has emerged as a
significant and serious problem in recent times, impacting both
Abdulhusein and Kadim/Archives of Ecotoxicology (2024) 28-36
35
humans and animals alike (Abdulhusein, and Caglayan 2022)
The success of the sensitivity test is influenced by various
factors, including the quantity and type of medium employed in
the experimental procedure. In this particular study, Mueller
Hinton medium was utilized, which is known for its ability to
support the growth of the bacteria being investigated.
Additionally, the pH constant value plays a significant role in
facilitating the effectiveness of the antibiotic being tested (Barry
1976). It is important to note that other factors, such as the size
of the inoculum used in the experiment and the incubation
conditions, also contribute to the success of the sensitivity test.
The findings of this study indicate that the bacteria exhibit a
notable sensitivity to the group of organic plant extracts, which
could possibly exceed their sensitivity to certain antibiotics, as
evidenced by the obtained results.
5. Conclusion
Over a period of time, many kinds of antibiotics have been
discovered. However, due to their uncontrolled usage, several
bacteria and fungi have developed resistance against these
antibiotics. Consequently, it is necessary that alternative
approaches be employed in our continuing struggle against
microorganisms. Over the past decade, there has been a
significant increase of Eichhornia crassipes in Iraq, particularly
in the southern regions of the Tigris River. This occurrence could
potentially elucidate the lack of attention given to river cleaning
efforts and the subsequent decrease in water levels. However, it
is worth considering the potential utilization of alcoholic
extracts of leaves from this plant as cost-effective alternative
medications for combating certain multidrug-resistant Gram-
negative and Gram-positive bacteria in controlled laboratory
environments, similar to findings from previous studies.
Additionally, an alternative explanation suggests that the
extracted materials possess a chemical affinity for interacting
with the components of the bacterial cell, facilitated by the
presence of receptors on the bacterial cell wall.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
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