As a result of antibiotic resistant microorganisms, infectious diseases remain one of the major causes of death. The rate at which microbial organisms continue to be resistant is significantly high globally (Schmitz et al., 1999). Consequently, the elevated level of resistance of pathogens and the ineffectiveness of the antibiotics has created a need to find other options (Ravikumar et al., 2010a). Manufacturing of new drugs, which are effective and without any other consequences is very necessary in order to deal with these issues. Overall, in order to come up with stronger antibiotics for killing the bacteria, viruses, fungi, and other harmful microorganisms, marine plants, such as mangroves, seaweeds, seagrasses, and marine sponges have been subjected to deep research (Ravikumar et al., 2009&2011). A medical plant has medical elements or substances that can be used for medical purposes: either it can be utilized as medicine, or it can be used to make a drug (Sofowora, 1982). Medicinal plants, over time, have played a significant role in curing human diseases and almost three quarters of the world’s population use plants to carry out health surveys (Farnsworth, 1994; Joy et al., 1998 and Harvey, 2000). Typically, natural products, as well as the newest drugs, are majorly made from plants and microbes (Hayashi et al., 1997; Armaka et al., 1999; Lin et al., 1999a &b; Basso et al., 2005 and Harvey, 2000). The bioassay-guided isolation is key in drug synthesis from the naturalproducts and is derived from the traditional uses of local plants (ethnobotanical and ethanopharmacological applications) (Atta-ur-Rahman and Choudhary, 1999). Seagrasses are marine plants which are found in large numbers in the tidal and sub tidal parts most sear apart from those in the Polar Regions. People who live in the coastal regions are well known for using the leafy part of seagrasses as food (Hemminga and Duarte, 2000). Seagrasses have been widely used as medicine to various ailments, including skin problems, fiver, muscle pains, and stomach aches, among other ailments in folk medicine (de la Torre-Castro and Rönnbäck, 2004). Seagrasses were also famous in India for managing heart conditions, nutritious purposes, fertilizers, as well as animal feeds (Newmaster et al., 2011). A number of seagrasses have been highly associated with antibacterial activities. For instance, ​Halophila stipulacea, Cymodocea serrulata ​and ​Halodule pinifolia ​(Kannan et al., 2010). The genotoxicity assays based on molecular techniques which have been exploited. Random amplified polymorphic DNA (RAPD) .

1. As a result of antibiotic resistant microorganisms, infectious
diseases remain one of the major causes
of death. The rate at which microbial organisms continue to be
resistant is significantly high globally
(Schmitz et al., 1999). Consequently, the elevated level of
resistance of pathogens and the
ineffectiveness of the antibiotics has created a need to find
other options (Ravikumar et al., 2010a).
Manufacturing of new drugs, which are effective and without
any other consequences is very
necessary in order to deal with these issues. Overall, in order to
come up with stronger antibiotics for
killing the bacteria, viruses, fungi, and other harmful
microorganisms, marine plants, such as
mangroves, seaweeds, seagrasses, and marine sponges have
been subjected to deep research
(Ravikumar et al., 2009&2011).
A medical plant has medical elements or substances that can be
used for medical purposes: either it
can be utilized as medicine, or it can be used to make a drug
(Sofowora, 1982). Medicinal plants, over
time, have played a significant role in curing human diseases
and almost three quarters of the world’s
population use plants to carry out health surveys (Farnsworth,
1994; Joy et al., 1998 and Harvey,
2000). Typically, natural products, as well as the newest drugs,
are majorly made from plants and
microbes (Hayashi et al., 1997; Armaka et al., 1999; Lin et al.,
1999a &b; Basso et al., 2005 and
Harvey, 2000). The bioassay-guided isolation is key in drug
synthesis from the naturalproducts and is

2. derived from the traditional uses of local plants (ethnobotanical
and ethanopharmacological
applications) (Atta-ur-Rahman and Choudhary, 1999).
Seagrasses are marine plants which are found in large numbers
in the tidal and sub tidal parts most
sear apart from those in the Polar Regions. People who live in
the coastal regions are well known for
using the leafy part of seagrasses as food (Hemminga and
Duarte, 2000). Seagrasses have been widely
used as medicine to various ailments, including skin problems,
fiver, muscle pains, and stomach aches,
among other ailments in folk medicine (de la Torre-Castro and
Rönnbäck, 2004). Seagrasses were also
famous in India for managing heart conditions, nutritious
purposes, fertilizers, as well as animal feeds
(Newmaster et al., 2011). A number of seagrasses have been
highly associated with antibacterial
activities. For instance, Halophila stipulacea, Cymodocea
serrulata and Halodule pinifolia (Kannan et
al., 2010).
The genotoxicity assays based on molecular techniques which
have been exploited. Random amplified
polymorphic DNA (RAPD) assay based on PCR amplification of
random DNA fragments of genomic
DNA (Atienzar et al., 1999). It has been used to detect DNA
damage and mutations in different
organisms (Savva, 1998 and Atienzar et al., 2001)
The present study was undertaken to investigate the
antibacterial activity of seagrass, Halodule
uninervis from the Jeddah city (Obhur Aljanoubiyah) in Saudi
Arabia against some pathogenic
bacteria.

3. Materials and Methods
Sample collection
Fresh leaves of Halodule uninervis were collected from the
intertidal region of Jeddah city (Obhur
Aljanoubiyah) 21º 42' 32.2'' N 39º 05' 47.3'' E (Lat.) then,
rapidly attended to the laboratory in sterile
plastic bags containing water to inhibit the evaporation.
Extraction
According to Boreu and Derevici, (1978), ten Grams of dried
seagrasses leaves samples. Adding 100 ml
of distilled water or organic solvents (Ethanol, Ethyl acetate,
Chloroform and Petroleum ether) (1:10
W/V) to make the extraction by using separating funnel and
shaking for 72 hours at room
temperature. Their solvents extract was filtered through
Whatman filter paper (No.1) and then
evaporating the solvents under low pressure at 40°C until
dryness. The plant extracts were all
dissolved in DMSO and kept in small closed vials at low
temperature 4°C.
Bacterial Strains
Seven tested bacterial strains were (four Gram-positive:
Bacillus subtilis (ATCC11774);
Methicillin-Resistant Staphylococcus aureus (MRSA)
(ATCC977); Staphylococcus aureus (ATCC29213)
and Micrococcus luteus (ATCC4698) and three Gram-negative:
Escherichia coli (ATCC8739); Klebsiella
pneumoniae (ATCC700603) and Pseudomonas aeruginosa

4. (ATCC27853). Those strains were provided
by Microbiologics® USA. The bacteria were obtained from
King Abdulaziz Hospital, Jeddah, Saudi
Arabia.
Antibacterial Activity
Antibacterial activities of plant extracts were tested against
different test microorganisms using agar
well diffusion method described by Egorove (1985). Pouring 20
ml of Mueller-Hinton agar in petri
dishes. A suspension of testing microorganisms were add to
Mueller-Hinton agar for bacteria. Using
sterile cork borer, three wells of 5 mm diameter in agar plate
were made. Putting 50 μl of the tested
leaves extracts in each well. Plates were left for one hour at 4°C
and then incubated for 24 h at 37°C.
Inhibition zones (including the diameter of disc) were
measured. The obtained results were compared
with DMSO as a negative control and with different antibiotic
as a positive control.
DNA Extraction
Genomic DNA was extracted from bacterial samples using
QIAamp DNA Mini Kit (QIAGEN, USA)
according to manufacturer Extracted DNA was stored at -20ºC
until further use.
Results
All four tested solvents extracts was inhibited the growth of all
bacterial pathogens by different zones.
The highest zone of inhibition by ethanolic extract against P.
aeruginosa was showed (33.33 mm)

5. followed by (24.67 mm) with B. subtilis in the same extract.
The lowest zone of inhibition by
petroleum ether extract against S. aureus was obtained (11.67
mm). No inhibition zone was seen by
distilled water except in P. aeruginosa which was appeared
(14.67 mm) zone. The antibacterial
activity of Halodule uninervis extracts on seven bacterial
pathogens were presented in Table 2.
Molecular RAPD-PCR of Pseudomonas aeruginosa
Twelve preselected random primers (RAPD) exhibited
polymorphism obtained from the DNAs of
seven samples of PC: Pseudomonas aeruginosa Control, PT:
Pseudomonas aeruginosa Treatment.
Study P. aeruginosa under investigation was the DNA based
analysis of plant extraction treatment. All
primers used in the present study resulted in the appearance of
PCR products with varied fragment
numbers as shown in (Figure 1). A total of 102 DNA fragments
were detected across the twelve
random primers, 37 of them were polymorphic (about 36%).
Primer UBC-208 has 8 amplicons which included 3 polymorphic
amplicons and 5 monomorphic
fragments that shows 60% percent of polymorphic. The
fragment sizes ranged from 500 to 1500bp
(Figure 1.A). Primer UBC-228 has 12 amplicons which included
7 polymorphic amplicons and 5
monomorphic fragments that shows 71% percent of
polymorphic. The fragment sizes ranged from
400 to 2000bp (Figure 1.B). Primer UBC-241 has 12 amplicons
which included 6 polymorphic
amplicons and 6 monomorphic fragments that shows 100%
percent of polymorphic. The fragment

6. sizes ranged from 300 to 2000bp (Figure 1.C). Primer UBC-270
has 3 amplicons which included 0
polymorphic amplicons and 3 monomorphic fragments. The
fragment sizes ranged from 300 to 900bp
(Figure 1.D).
Primer UBC-272 has 5 amplicons which included 2 polymorphic
amplicons and 3 monomorphic
fragments that shows 66% percent of polymorphic. The
fragment sizes ranged from 400 to 1500bp
(Figure 1.E). Primer UBC-275 has 8 amplicons which included
3 polymorphic amplicons and 5
monomorphic fragments that shows 60% percent of
polymorphic. The fragment sizes ranged from
400 to 6000bp (Figure 1.F). Primer UBC-277 has 8 amplicons
which included 2 polymorphic amplicons
and 6 monomorphic fragments that shows 33% percent of
polymorphic. The fragment sizes ranged
from 400 to 4000bp (Figure 1.G). Primer UBC-287 has 6
amplicons which included 1 polymorphic
amplicons and 5 monomorphic fragments that shows 20%
percent of polymorphic. The fragment sizes
ranged from 300 to 3000bp (Figure 1.H).
Primer UBC-325 has 6 amplicons which included 2 polymorphic
amplicons and 4 monomorphic
fragments that shows 50% percent of polymorphic. The
fragment sizes ranged from 300 to 3000bp
(Figure 1.I). Primer UBC-327 has 15 amplicons which included
9 polymorphic amplicons and 6
monomorphic fragments that shows 60% percent of

7. polymorphic. The fragment sizes ranged from
300 to 2500bp (Figure 1.J). Primer UBC-700 has 13 amplicons
which included 0 polymorphic amplicons
and 13 monomorphic fragments. The fragment sizes ranged from
300 to 2500bp (Figure 1.K). Primer
UBC-44 has 6 amplicons which included 2 polymorphic
amplicons and 4 monomorphic fragments that
shows 50% percent of polymorphic. The fragment sizes ranged
from 300 to 2000bp (Figure 1.L).
Discussion
Due to the increasing of the resistance rate of microorganisms
on the antibiotic drugs in the recent
past, the field of clinical treatment for infectious diseases has
faced big problems diseases (Ravikumar
et al., 2010).There are various reports on the ability of
seaweeds, mangroves and other marine plants
to kill microorganism activities while little has been written
about the global seagrasses and there is
also minute information about those (Kannan et al., 2010). The
purpose of this study is to assess and
compare how and the potential of seagrass extracts in the
synthesis of bioactive substances, which
can be used for therapeutic purposes. The exhibition of
antimicrobial activities in the seagrass
demonstrated their ability to produce bioactive secondary
metabolites.
The antibacterial activity of three different leaves extracts of H.
uninervis against seven bacterial
pathogens strains were effective. Among them, ethanol extract
was the more effective against P.
aeruginosa than other extracts, this showed that ethanol is
suitable for extracting active compounds
from seagrass. These findings were supported by the earlier

8. studies which suggested that the
methanolic extract of Enhalus acoroides produced a stronger
effect against P. aeruginosa, K.
pneumoniaeand S. aureus when compared to the hexane extract
(Alam et al., 1994). This study
demonstrated that the best antimicrobial activity was in the
ethanoic extract, which concurred with
other earlier reports (Umamaheshwari et al., 2009) and the
ethanolic and methanolic extractions of
the seagrasses Halophila ovalis and Halodule pinafolia were
preferable than the other tested extracts
because their better inhibition zones against tested bacteria
(Mani et al., 2012).
Our most recent study showed that Gram-negative bacteria were
more imoressible compared to the
Gram-positive bacteria. This concurred with the findings of a
certain report that there was an
anti-fouling of various marine organisms against Bacillus and
Pseudomonas sp. (Bhosale et al., 2002).
The inconsistencies of the extracts in the antibacterial activity
can be as a result of the variations of
antimicrobial agents from species to species (Lustigman and
Brown, 1991).
The lowest antimicrobial effect was observed in the extracts of
ethyl acetate of H. uninervis. Also, the
results suggested that P. aeruginosa had a moderate sensitivity
rate when the aqueous extracts were
used against them, unlike the other microorganisms. This agrees
with another report that aqueous
extracts of C. routundata did not show any activity with all the
test bacteria which showed very poor
activity (Mani et al., 2012).
Also because of the reported study of phytochemical analysis of

9. Hexane, Chloroform, Ethyl acetate,
Ethanol and Aqueous extracts of three glycosides have saponins
and tannins. Sugars and quinine were
absent in all the three seagrasses (Cymodocea serrulata,
Halophila ovalis and Halodule pinifolia)
(Sangeetha and Asokan, 2016). The earlier reports like Ergene
et al., (2006) who revealed the
presence of tannins, saponins, proteins, resins, reducing sugar,
acidic compounds, alkaloids, cardiac
glycosides and terpenoids in the phytochemical analysis of C.
rotundata. Glycoside, saponins, tannins,
flavonoids, terpenoides and alkaloids which considered as
phytochemical compounds
In the RAPD-PCR profile, the change of fragments number and
the variation in their intensity due to
the change of genetic material. The stability of genomic
template (GTS, %) refers to the extent of
damaged DNA and DNA efficiency to repair and replication
(Rocco et al., 2011). For instance, the high
level of damaged DNA dose not reduce the stability of genomic
template that due to the inhibition of
DNA repair and replication resulting of excessive or lethal
actions of the plant extract.
The results show a clear evidence of plant extract ability to
mutate which appearance of many genetic
fragments compared with untreated bacteria (control). These
genetics techniques obtained by (Adam
et al., 2000; Morita et al., 2005 and Gilani et al., 2006).

10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973789/#CR31