ANTIBACTERIAL ACTIVITY OF MICROORGANISMS ASSOCIATED WITH MARINE INVERTEBRATES FROM THE MOROCCAN ATLANTIC COAST

Mohammed El Amraoui 1,2 , Meriem Tarbaoui 3, *, Majida El Wahidi 1 , Aziz Fassouane 1,4 , Belkassem El Amraoui 1,5 and Toufiq Bamhaoud 1 . 1. Laboratoire Controle Qualite en Bioindustrie et Molecules Bioactives, Faculte des Sciences, Universite Chouaib Doukkali, BP 20, 24000 El-Jadida, Maroc. 2. Laboratoire du Maghreb, BP 2863, Rabat, Maroc. 3. Laboratoire Biomolecules et Synthèse Organique, Faculté des Sciences Ben M’sik, Universite Hassan IICasablanca, B.P 7955 Casablanca, Maroc. 4. Ecole nationale de commerce et de gestion, BP 122 El-Jadida, Maroc. 5. Physicochimie des Milieux Naturels et Molécules Bioactives (PMNMBA), Faculté Polydisciplinaire de Taroudant, Université Ibn Zohr, BP 271, 83000 Taroudant, Maroc. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In vitro antibacterial screening of bacterial strains isolated from marine invertebrates, collected from the Moroccan Atlantic coast, against human pathogenic bacteria was conducted in this study. However fourteen marine microorganisms were tested against three Gram-positive bacteria (Staphylococcus aureus, Bacillus sp. and Enterococcus faecalis) and three Gram negative bacteria (Escherichia coli, Pseudomonas fluorescens and Pseudomonas sp.) using the agar disk-diffusion and the agar cylinders methods. The identification of the isolates shows that are belong to the genera Enterobacter, Morganella, Aeromonas, Pantoea, Kluyvera, Raoultella, Stenotrophomonas, Pseudomonas, Sphingomonas and Staphylococcus which indicates a diversification in the marine microflora. The evaluation of antibacterial activity of these isolates on human pathogenic bacteria shows that all strains are active against at least one pathogen studied for the two used methods. For the agar cylinders method, six isolates are showing a significant antibacterial activity with inhibition zone diameters greater or equal to 15 mm (Aeromonas sobria, Enterobacter cloacae, Stenotrophomonas maltophilia, Staphylococcus capitis, Pantoea sp3 and Raoultella ornithinolytica). For the agar disk-diffusion method, among 28 extracts of the marine isolates, five extracts which are active against two pathogenic bacteria (Pseudomonas sp and Enterococcus faecalis) with inhibition zone diameters greater or equal to 15 mm (two ethanol extract of Enterobacter cloacae , ethanol extract of Raoultella ornithinolytica, ethanol extract of Morganella morganii and ethanol extract of Aeromonas sobria). By comparing the two used methods we can conclude that the agar cylinders method gave better results compared to the agar disk-diffusion method.
Isolation and identification of microorganisms:-1 cm 3 of each marine organism was sampled using a sterile scalpel, ground in a sterile mortar in the presence of sterile physiological water, and inoculated on the culture media. After inoculation, the dishes are incubated in an oven at 35 ± 2 ° C for 24 hours. Several subcultures were conducted to obtain pure culture.
The identification of marine microorganisms was carried out at the Maghreb laboratory, Rabat (Morocco) to the following protocol: the Gram-positive or Gram-negative bacterial differentiation was carried out by the Gram staining. The Gram-positive bacteria are tested for catalase whereas Gram-negative bacteria undergo the oxidase test, and there-after, according to the results of biochemical tests, a type of gallery is used. Galleries ''ID32 STAPH'' are used for Gram (+), catalase (+), galleries ''ID32 STREP'' for Gram (+), catalase (−). Whereas galleries ''ID32 GN'' for Gram (−), oxidase (+) and galleries ''ID32 E'' are used for the Gram (−), oxidase (−). Galleries and results are analyzed using an identification device ''ATB Expression 2000 SYSTEMS bioMerieux VITEK'' linked to the computer.
Antibacterial activity of isolated microorganisms:-Antibacterial activity of marine microorganisms was estimated by the agar cylinders and the agar disk diffusion methods against three Gram-positive bacteria (Staphylococcus aureus ATCC25923, Bacillus sp. CIP104717 and Enterococcus faecalis ATCC19433) and three Gram-negative bacteria (Escherichia coli CIP54127, Pseudomonas fluorescens CAN 228-1 and Pseudomonas sp.) using Mueller-Hinton agar medium [bioMérieux® SA]. These 1129 reference strains were obtained from the Collection of Pasteur Institute (CIP), and from the American Type Culture Collection (ATCC). The tests pathogens inoculate were prepared by suspending in 10 mL of sterile water the colonies from 18 h culture on Luria Bertani medium. The cell density was determined by a haemocytometer and adjusted to10 6 UFC/mL.

Agar disk diffusion method:-
The isolated bacteria were cultured in nutrient broth for 7 days then each culture was extracted with ethyl acetate (3 × 100 ml). The ethyl acetate extracts were combined, dried on anhydrous sodium sulphate (Na 2 SO 4 ), filtered and concentrated at reduced pressure to give an ethyl acetate extract (extract C). The aqueous phases were lyophilised and twice dissolved in absolute ethanol, then filtered and concentrated at reduced pressure to give an ethanol extract (extract B).The extracts (B and C) were tested for their antibacterial activity using agar disk diffusion methods.
6 mm diameter cellulose discs were saturated with 300 µg of extract (B or C) of isolated bacteria then applied on the test media which were previously inoculated with each pathogen strain. Plates were first kept at 4 °C for at least 2 h to allow the diffusion of chemicals, and then incubated at 37 °C. Inhibition zones were measured after 24 h of incubation. Standard disks of the antibiotic tetracycline (30 µg) and penicillin (10 µg) served as the positive antibacterial controls. All tests were performed in triplicate.

Agar cylinders method:-
Isolates were grown on marine agar plates for four days at 37 °C, and then a calibrated cylinder (6 mm in diameter) was cut out and placed on the surface of the test medium (Muller Hinton) previously inoculated with each test strain. Plates were kept first at 4 °C for at least 2 h to allow the diffusion of active metabolites, and then incubated at 37°C. Inhibition zones were measured after 24 h of incubation.

Results and Discussion:-
Six microorganisms were identified from the sponge Ircinia spinulosa (Sarcotragus), four microorganisms from the sponge Paraleucilla magna, one microorganism from the sponge Cliona viridis, one microorganism from the sponge Haliclona viscosa and tow microorganisms from the sea cucumber. The results of identification of microorganisms are summarized in table 1. Identified isolates belong to the genera Enterobacter, Morganella, Aeromonas, Pantoea, Kluyvera, Raoultella, Stenotrophomonas, Pseudomonas, Sphingomonas and Staphylococcus. This shows a diversification in the marine microflora. Isolates species and there antibacterial activity against six pathogen bacteria were reported in tables 2 and 3.
For the agar cylinders method (Table 2), the results show that all strains (14 isolates) have antibacterial activity against at least one out of six test pathogens studied.
Four microorganisms of the genus Enterobacter are of the same species (Enterobacter cloacae). Three of them, Cv, Is 4 and Pm 4 , were isolated from marine sponges (Cliona viridis, Ircinia spinulosa, Paraleucilla magna) while Sc 1 was isolated from Sea cucumber. The four isolates show significant antibacterial activity but only Sc 1 and Pm 4 haven't antibacterial activity against Pseudomonas fluorecens. Two microorganisms of the genus Aeromonas are of the same species (Aeromonas sobria). One of them, Sc 2 , was isolated from Sea cucumber while Is 1 was isolated from marine sponge Ircinia spinulosa. The two isolates show significant antibacterial activity but only Sc 2 haven't antibacterial activity against Pseudomonas fluorecens. This strain has a very high potential resistance against the antibacterial action of the majority of microorganisms. For the agar disk-diffusion method (Table 3), the results show that all isolate have antibacterial activity against at least one out of six test pathogens studied. All B extracts (14 extracts) of studied marine microorganisms have displayed some activity against at least one out of six test pathogens studied.
Except two C extracts of isolates (Kluyvera cryocrescens and Sphingomonas paucimobilis) were not active against any pathogens studied.

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Among 28 extracts of the marine microorganisms, five extracts which are showing a significant antibacterial activity with inhibition zone diameters greater or equal to 15 mm:  four extracts were active against Pseudomonas sp, two B extract of Enterobacter cloacae (Is 4 and Cv), B extract of Raoultella ornithinolytica (Is 5 ) and B extract of Morganella morganii (Hv);  four extracts (B extracts of Aeromonas sobria (Is 1 ), Enterobacter cloacae (Is 4 and Cv) and Raoultella ornithinolytica (Is 5 )) were active against Enterococcus faecalis.
By comparing the two methods used to determine the antibacterial activity of the studied microorganisms against the six test pathogens studied, we can conclude that the agar cylinders method gave better results compared to the agar disk-diffusion method.
It is evident then that marine macro-and microorganisms, living in an environment where competition and predation are the maximum without physical defence structure, defends themselves by producing chemicals to survive. It is found that the sea surface or cavum of marine organisms such as seaweeds and invertebrates are more nutritious than inanimate material and seawater, and a large number of bacteria could live on it (Sponga et al., 1999). These bacteria species are not generally real symbiotic to the host, but they can instead be regarded as associated bacteria (Ponce et al., 1999), and are forced to develop resistance to antibiotics secreted by its host. In fact, they can produce antibiotics and antifungal to inhibit or limit the development and growth of other competitive microorganisms. It is evident then that the antibacterial spectra of the active marine bacteria are different (Zheng et al., 2005). This difference comes from the variation of the ecological conditions of the environment of the microorganisms (El-Wahidi et al., 2011). However, some isolates of the same species show a difference in their antibacterial activity. Moreover, molecular studies of these species could provide the answer.

Conclusion:-
Preliminary results show that marine microorganisms associated to different marine invertebrates have an antibacterial potential. They can also constitute a potential source of new compounds to be used in the field of health. Aeromonas sobria (Is 1 ), Enterobacter cloacae (Is 4 and Cv), Stenotrophomonas maltophilia (Is 6 ), Staphylococcus capitis (Pm 3 ), Pantoea sp3 (Is 2 ) and Raoultella ornithinolytica (Is 5 ) bacteria that exhibit a significant antibacterial activity should be investigated for isolation and identification of natural antibacterials.