As a result of antibiotic resistant microorganisms, infectious diseases remain one of the major causes of death

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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) 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.

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 (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) 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 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 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 studies which suggested that the methanolic extract of ​Enhalus acoroides produced a stronger effect against ​P. aeruginosa, K. pneumoniae​and ​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 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).

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