2199-2211, 2010 © 2010, Insinet Publication

Distribution and Biodiversity of Antimicrobial Agents Producing Marine Bacteria along Alexandria Coasts

1El-bestawy, E.A.; 2Abou-elela, G.M.; 2El-shneway, M.A.; 3Gohar, Y.M. and 2Wefky, S.H.

1Institute of Graduate Studies and Research, Alexandria University, Egypt. 2National Institute of Oceanography and Fisheries, Alexandria, Egypt 3Botany Department, Faculty of Science, Alexandria University, Egypt.

Abstract: Water and sediment samples were collected seasonally from nine stations along Alexandria coasts to determine the distribution of the bacterial groups which were able to produce antimicrobial agents and studying the correlations between their counts and the prevailing physico-chemical factors. Winter exhibited the highest total viable count in water and sediments while autum recorded the lowest counts. The counts of bioactive compounds producing bacteria exhibited different distribution pattern and these bacterial groups spread mainly during Spring . El-Shatby harboured the highest total viable count, but Abu-Qir had the lowest counts. All bacterial isolates were tested for their activity spectra against different pathogens (S.aureus, S.faecalis, P. aeruginosa, M.luteus, E. coli and C.albicans). Only 34 strains showed antimicrobial activity. Numerical taxonomy was represented in clustering of these strains . Gram positive clustered at 90% similarity level into 3 phena and included Enterococcus sp., Arthrobacter sp., Micrococcus sp., Corynebacterium sp., and Bacillus sp. Gram negative clustered at 94% and identified as Flavobacterium sp., Moraxella sp., Acinetobacter sp.

Key words: Marine bacteria- bioactive compounds-Identification Alexandria Coasts

INTRODUCTION MATERIALS AND METHODS

Marine macroorganisms and microorganisms 2.1.Study area: Water and sediment samples were produce a dizzying array of active compounds collected from 9 sites along Alexandria coasts, including terpenes, steroids, peptides and alkaloids, extended from Abu-Qir until El-Agami as shown in which have potent activities as anti-microbial agents. (Fig. 1). Some compounds isolated from marine macroorganisms have been shown to be of microbial origin [1,2] 2.2.samples Collection and Preservation: Samples During the past two decades, research on marine were collected seasonally from Spring 2004 to Spring bacteria has highlighted the tremendous potential of 2005 according to the standard procedures as described these microorganisms as source of new bioactive by the International Organization for Standardization [7]. secondary metabolites [3,4]. Many marine heterotrophic Water samples were collected at 25-35 cm below the bacteria are known to produce antibacterial substances sea water surface and sediment samples were collected [5]. Many free living and sediment-borne marine using a piston corer. Samples were sent to the bacteria have been shown to produce secondary laboratory and examined within 2 to 3 hours of metabolites that display antibacterial properties, and it collection. has also been reported that particle-attached marine bacteria are more likely to produce inhibitory 2.3.physico-chemical Analysis: Water temperature was compounds than free living bacteria [6]. measured by a normal thermometer (0- 50oC). pH was The present study aimed to survey the distribution determined using Beckman digital portable pH-meter of bio-active compounds producing marine bacteria model 3650. Salinity, dissolved oxygen, oxidizable along the sea shores of Alexandria and the organic matter in water and organic matter contents in environmental factors affecting their distribution, in sediments were determined according to APHA[8]. addition to the identification of the isolates. Ammonia was determined according to IOC [9]. Nitrites

Corresponding Author: Dr. Gehan M. Abou Elela, Associate professor, Marine Microbiology laboratory, Environmental Division, National Institute of Oceanography and Fisheries, Qaiet Bay, El-Anfushy, Alexandria Egypt. Tel: 0020128636344 2199 J. Appl. Sci. Res., 6(12): 2199-2211, 2010

were determined according to Grasshoff [10]. While 2.8. Statistical analyses: Simple correlation coefficient nitrates and phosphates were determined as described was applied to asses the relationships between bacterial by Strickland and Parsons[11]. counts and the different physico-chemical parameters. Data were analyzed according to Steel and Torrie [19]. 2.4. Microbial indicators: Different pathogenic strains such as S. aureus (ATCC 6538), S. faecalis (ATCC RESULTS AND DISCUSSION 8043), P. aeruginosa (ATCC 8739), E. coli (ATCC 8739), M. luteus (ATCC 10240) and C. albicans were 3.1.physico-chemical Characteristics of the Studied used as indicator strains. Areas: The results in Table (1) show that, Abu-Qir water recorded the lowest annual mean for temperature 2.5. Bacteriological Analyses: Total viable count o (TVC) was determined on aged sea water agar and the and pH (23.20 C and 7.90). Sidi-Bishr achieved the plates incubated at 30 oC for 24 hrs [8]. The highest annual mean for salinity (38.20‰) but recorded antimicrobial activity against the different microbial the lowest annual means for ammonia and nitrite (1.69 indicators as tested by the double-layer technique used and 7.45 µg/l). Stanley recorded the highest annual for studying bio-active compounds production [12]. The mean for organic matter (159.39 mgO2/l) while El- antibiotic-producing colonies showed a clear zone of Shatby recorded the highest annual mean for pH. Kaiet inhibition. The grown colonies were counted separately. Bay harboured the lowest annual means for nitrate The counts of each type was then compared with the concentrations and organic matter (11.20 µg /l and total number of heterotrophs in the sample. 17.07 mgO2/l). El-Max drainage recorded the highest means for ammonia, nitrite, nitrate and phosphate 2.6.antimicrobial Activity Spectra of Bacteria (70.03, 38.59, 67.25 and 91.54 µg /l respectively). The Producing the Bioactive Compounds: Antibacterial lowest annual mean for phosphate detected in El-Agami activity was tested using the paper disk assay [13]. (25.67 µg /l). The chemical characteristic of sediments Suspension of each bacterial isolate was centrifuged at 13,000 rpm for 10 min. to harvest the bacterial cells. showed in (Table, 2). Abu-Qir recorded the highest 50 µl of the supernatants were used to saturate annual mean for organic matter (21.50 mg/g) while antibiotic assay disks (5 mm).Pathogenic indicator Sidi –Bishr detected the lowest annual mean for pH bacteria Staphylococcus aureus (ATCC 6538), (7.91). Stanley recorded the highest annual mean for Streptococcus faecalis (ATCC 8043), Pseudomonas nitrates (27.57 µg /g) and the lowest annual mean for aeruginosa (ATCC 8739), E. coli (ATCC 8739), nitrite (0.32 µg /g). El-Shatbi achieved the lowes Micrococcus luteus (ATCC 10240) and Candida annual mean for ammonia (0.88µg /g). The lowest albicans were swabbed onto the agar plates. The disks annual mean for pH (7.54) and the highest annual were then placed onto the agar surfaces and incubated mean for ammonia (13.99µg /g) in Agami. El-Mex at 37°C for 12 hours. The diameters of any inhibition annual mean for nitrite was the highest (5.85 µg /g). zone that formed around the paper disks were then El-Mex drainage detected the lowest phosphate annual measured. mean (5.44µg /g) while El-Agami recorded the highest annual means for phosphate and ammonia (25.44 and 2.7.phenotypic Characterization and Numerical 13.99 µg /g respectively). Analysis: Strains with antimicrobial activity (34 strains) were subjected to characterization morphologically and physiologically ( Gram reaction and spore formation, 3.2.viable Count Exhibiting Antimicrobial Activity in the pH range at values (5-9), temperature range Sea Water and Sediments: The total viable count (20_50oC), sodium chloride requirement (0-13%) ), and the count of different groups at each site are biochemical characters also studied such as: acid epresented in (Table 3, 4). production , Voges Proskauer test, indole production, citrate utilization , nitrate reduction, urease production, Antistaphylococcus: group was detected in all sites oxidase, catalase, glucose fermentation, gelatinase under investigation but disappeared in Kaietbay. Spring production , sulphate precipitation and antibiosis against was the favorite season for its spread. The highest [14] different pathogens . count was achieved in El-Montazah (108 CFU/ml) while The characters of each strain were coded as the lowest count was detected in Sidi-Bishr (103 negative (0) or positive (1). The simple matching [15] [16] CFU/ml) during spring 2004.Considering sediments, the coefficient (SsM) and the Jaccard coefficient (Sj) highest record was that detected in El-Mex during were used and clustering was achieved by Unweighted spring 2005 (16x107 CFU/g) while the lowest count Pair Group Average linkage UPGMA [17]. The was detected in El-Montazah during spring 2005 computations were performed by using SYSTAT-PC 3 program V7 [18]. on an IBM computer. (16x10 CFU/g)

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Antistreptococcus: group was detected in all sites disappeared during summer in all sites. The highest except for El-Mex Drainage. Spring was the suitable counts recorded in El-Montazah during winter (2x108 season for its spread. The highest count was recorded CFU/ml) followed by El-Shatby during spring 2005 in El-Agami during winter (2x108 CFU/ml). The lowest (6x107 CFU/ml) and Stanley (5x107 CFU/ml). The counts were detected during autumn in the same region lowest record was that detected in Sidi-Bishr during (104 CFU/ml). In the sediments, it detected in all sites spring 2004 (103 CFU/ml) .It detected in sediments of except for El-Mex Drainage. The highest count was in all sites under investigation except for El-Mex Kaietbay during winter (108 CFU/g) and also the Drainage. It was found only in Abu-Qir and El-Mex highest annual mean (2x107 CFU/gm) and the lowest during winter where the highest counts were achieved count was detected in El-Montazah during spring 2005 (2x108 and 108 CFU/g) .High count was also achieved (104 CFU/g). in El-Mex during spring 2005 (108 CFU/g). The lowest count was that recorded in El-Montazah (9x103 CFU/g). Antipseudomonas: group was detected in all sites during spring except for Kaietbay and El-Mex 3.3. Statistical analyses: Simple correlation coefficient Drainage. The highest count was detected in El-Agami was performed to estimate the correlations among TVC (3x108 CFU/ml ) while the lowest count were detected of bacteria, counts of different bacterial groups in El-Montazah and El-Mex during spring 2004 and in producing bioactive compounds and the El-Agami during spring 2005 (2x105 CFU/ml). The physicochemical conditions prevailing in the studying highest count in sediments was detected in El-Shatby areas. (109 CFU/g) during winter representing (27.77%) of the The total viable count positively correlated with total viable count, it also represented the highest annual salinity and organic matter during winter and spring mean( 2x108 CFU/gm). On the other hand, the lowest 2005 respectively. detection was recorded in El-Montazah during spring Antistaphylococcus group in water during spring 2005 (2x104 CFU/g). 2005 was positively correlated with ammonia, nitrate and phosphate concentrations, also positively correlated Antimicrococcus: group distribution showed that, the with phosphate concentration during spring 2004. There highest record was that in El-Agami during winter (108 was a positive significant correlation between the same CFU/ml) The lowest records (105 CFU/ml) were in group and ammonia concentration in sediments during Abu-Qir during autumn and in Sidi-Bishr during spring spring 2005. 2005 . This group was absent in sediments of Abu-Qir There was a positive significant correlations and El-Mex Drainage. It was detected during spring between the counts of antipseudomonas group in only except for El-Shatby where it was detected during sediments and nitrate and nitrite concentrations during winter and recorded the highest count (108 CFU/g). The spring 2004 and winter. count decreased in El-Montazah during spring 2005 Antimicrococcus count in water was positively (12x103 CFU/g). The highest annual mean in El-Shatbi correlated with dissolved organic matter concentrations (2x107CFU/gm) and the lowest in El-Montazah (24x102 during autumn while their counts in sediments were CFU/gm). positively correlated with nitrate and phosphate during winter and spring 2004 respectively. Antiescherichia: group was detected only during spring Finally the counts of anticandida group in water and was dramatically absent during the other seasons. were positively correlated with nitrite concentration It disappeared completely in El-Shatby and Kaietbay all during autumn and in sediments were positively over the year. Sidi-Bishr achieved the lowest count (103 correlated with nitrate during spring 2005 and CFU/ml) in Spring 2004 ,while El-Mex and El-Mex phosphate concentration during winter. Drainage showed the highest records during Spring 2005 (107CFU/ml). In sediments, it was absent in El- 3.4. Antimicrobial Activity of the Bioactive Mex Drainage all over the year while it was detected Compounds Producing Marine Bacteria: As Shown in the other sites during spring and in Abu-Qir only in Table (5) ,thirty four isolates exhibited promising during autumn which recorded the lowest count (102 activity against 6 selected pathogens Sixteen strains CFU/g) .This group also appeared in Kaietbay and El- showed antagonistic effect at least against three

Mex during winter where the highest counts were pathogens while only two strains (S29 from kaietbay 8 achieved (2x10 CFU/gm).No detection was achieved and S14 from Sidi-Bishr) had broad spectrum effect during summer and autumn. against all tested organisms.

Anticandida: group spread in all sites during spring except for Kaietbay and Abu-Qir, while they

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Fig. 1: Alexandria Coast: The study area and sampling sites.

Table 1: Annual mean of the physicochemical parameters of sea water at the different sampling sites during the period of study.

Sampling sites Temp. (°C) pH Salinity (‰) Dis. oxygen Dis. NH4 Dis. NO2 Dis NO3 Total Dis PO4 Organic (ml O2/l) nirtrogen matter mgO2/l ------µg/l Abu-Qir 23.20 7.90 35.07 5.15 22.35 15.70 31.86 69.89 27.71 105 ------EL-Montazh 23.80 8.18 37.65 5.27 7.06 8.46 19.76 35.29 31.70 74.59 ------Sidi-Bishr 24.34 8.18 38.20 5.80 1.69 7.45 56.39 65.54 43.36 75.11 ------Stanley 24.04 8.14 36.06 7.50 2.15 8.97 22.19 33.33 34.81 159.39 ------El-Shatby 24.34 8.26 37.33 7.49 2.32 9.92 31.49 43.72 35.24 35.73 ------KaietBay 23.98 8.24 36.53 6.95 14.81 9 11.20 35.02 28.04 17.07 ------El-Mex 24.52 8.24 33.76 8.59 4.82 26.95 35.37 67.14 47.93 34.53 ------El-Mex Drainage 24.18 8.11 16.33 3.79 70.03 38.59 67.25 175.87 91.54 67.73 ------El-Agami 24.10 8.18 37.59 7.37 5.63 9.19 37.18 52.02 25.67 36.19

Table 2:Annual mean of the physicochemical parameters of sediments at the different sampling sites during the period of study.

Sampling sites pH Dis. NH4 Dis. NO2 Dis.NO3 Total nitrogen Dis.PO4 Organic matter ------µg/g mg/g Abu-Qir 7.66 9.47 0.73 13.76 24.10 20.52 21.50 ------El-Montazh 7.83 5.34 0.48 13.79 24.72 12.69 7.23 ------Sidi-Bishr 7.91 4.81 0.69 25.61 31.11 12.55 6.85 ------Stanley 7.84 5.46 0.32 27.57 33.35 7.69 14.43 ------El-Shatby 7.84 0.88 0.52 15.78 25.18 7.26 2.31 ------Kaietbay 7.54 9.77 0.65 11.99 22.41 9.27 5.65 ------El-Mex 7.68 3.37 5.85 10.62 19.48 23.79 17.17 ------El-Mex Drainage 7.58 8.73 0.47 11.21 20.43 5.44 15.58 ------El-Agami 7.79 13.99 1.41 20.61 28.76 25.44 8.39

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Table 3: Total viable count of bacteria and count of bacteria exhibiting antimicrobial activity (CFU/ml) isolated in sea water of the selected sites Sampling site Season TVC Anti-a* anti-b* anti-c* anti-d* anti-e* anti-f* Sampling Sites Abu-Qir Spring 04 16x106 106 00000 ------Summer 75x106 106 2x106 0000 ------Autumn 3x106 000 105 0 3x105 ------Winter 56x107 0 0 2x107 000 ------Spring 05 5x107 00107 106 5x106 0 ------Annual mean 108 4x105 4x105 6x106 22x104 106 6x104 El- Montazah Spring 04 15x106 105 2x105 2 x105 3 x105 2 x105 3 x105 ------Summer 3x108 000000 ------Autumn 7x107 000000 ------Winter 4x109 108 0 0 0 0 2 x108 ------Spring 05 2x107 0 3x106 2x106 106 106 ------Annual mean 9 x108 2x107 64x104 4x104 46x104 24x104 4x107 Sidi-Bishr Spring 0423x104 103 000103 103 ------Summer 6x107 2x106 00000 ------Autumn 5x107 000000 ------Winter 27x108 000000 ------Spring 05 107 105 8 x105 9 x105 105 105 2 x105 ------Annual mean 6 x108 42 x104 16x104 18x104 2x104 2x104 4x104 Stanley Spring 0424x106 106 00 106 0106 ------Summer 65x107 000000 ------Autumn 35x106 0 0 0 2x106 00 ------Winter 31x107 2x107 0 0 0 0 5 x107 ------Spring 05 4x107 3x106 106 2 x106 106 106 2 x106 ------Annual mean 2 x108 48x105 2x105 4x105 8x105 2x105 107 El-Shatby Spring 0412x106 000000 ------Summer 18x108 000000 ------Autumn 3x106 000000 ------Winter 39x109 000000 ------Spring 05 62x107 107 8 x107 3 x107 0 0 6 x107 ------Annual mean 8 x109 2x106 16x106 6x106 0 0 12x106 Kaietbay Spring 04 2x107 000000 ------Summer 6x107 000000 ------Autumn 1x106 000000 ------Winter 34x108 002 x108 000 ------Spring05 2x108 0107 0 4x107 00 ------Annual mean 7x108 0 0 4x107 000

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Table 3: Continue El-Mex Spring 04 17x106 2x105 0 2 x105 105 2 x105 2 x105 ------Summer 24x107 000000 ------Autumn 15x104 0 2x104 0000 ------Winter 36x108 0108 3 x108 000 ------Spring05 32x107 107 4 x107 4 x107 2 x107 107 3 x107 ------Annual mean 8x108 2x106 28x106 68x106 4x106 2x106 6 x106 El- El-Mex Spring 04 7x105 000000 Drainage ------Summer 39x107 000000 ------Autumn 104 000000 ------Winter 37x107 00108 000 ------Spring 05 55x107 4 x107 000107 107 El-Agami Annul mean 3x108 8x106 0 2x107 0 2x106 2x106 ------Spring 04 56x104 000000 ------Summer 245x105 00000p ------Autumn 3x105 0 104 000104 ------Winter 24x108 0 2 x108 3x108 108 0 ------Spring 05 23x105 3 x105 7 x105 2 x105 2 x105 6 x105 2 x105 Annual mean 5x108 6x104 4x107 6x107 2x107 12x107 42x103 a*= S.aureus, b*=S.faecalis, c*=P.aeruginosa, d*= M.luteus, e*= E.coli, f*= C.albicans and 0= no count

Table 4: Total viable count of bacteria and count of bacteria exhibiting antimicrobial activity(CFU/gm) in sediments of the selected sites . Sampling Sites Season TVC anti-a* anti-b* anti-c* anti-d* anti-e* anti-f* Abu-Qir Spring 04 32x105 0 0 0 0 0 105 ------Summer 7x108 000000 ------A utumn 4x103 000 1020 ------Winter 35x108 0 0 0 0 0 2x108 ------Spring 05 54x107 2x107 2 x107 ------Annual mean 9x108 0 4x106 0 0 4x106 4x107 El-Montazah Spring 04 3x107 000000 ------Summer 68x107 000000 ------Autumn 14x103 000000 ------Winter 33x108 000000 ------Spring 05 8x104 16x103 104 2x104 12 x103 5 x103 9 x103 ------Annual mean 8x108 32x102 2x103 4x103 24x102 103 18x102 Sidi-Bishr Spring 04 12x106 105 105 2x105 105 2x105 0 ------Summer 64x107 3 x107 00000 ------Autumn 104 000000 ------Winter 34x108 000000cc ------Spring 05 108 5x106 13 x106 6 x106 17 x106 107 6 x106 ------Annual mean 8 x108 7x106 26x105 106 17x106 2x106 106

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Table 4: Continue Stanley Spring 04 11x106 000000 ------Summer 45x107 107 00000 ------Autumn 21x103 000000 ------Winter 29x108 008 x108 000 ------Spring 05 8x107 5 x106 12 x106 7 x106 4 x106 8 x106 ------Annual mean 7 x108 2x106 106 16x107 106 8x105 16x105 El-Shatby Spring 04 2x106 3x105 2 x105 4 x105 2 x105 105 3 x105 ------Summer 1x108 000000 ------Autumn 5x103 000000 ------Winter 36x108 00109 108 00 ------Spring 05 24x107 6x107 6 x107 2 x107 3 x107 4 x107 ------Annual mean 8 x108 107 4x104 2x108 2x107 6x106 8x106 Kaietbay Spring 04 35x105 000000 ------Summer 31x107 000000 ------Autumn 104 000000 ------Winter 26x108 0108 0 0 2x108 0 ------Spring05 9x107 3 x106 12 x106 107 9 x106 107 ------Annual mean 5 x108 6x105 2x107 2x106 2x106 42 x106 2x106 El-Mex Spring 04 19x106 2x106 0 0 106 0 105 ------Summer 4x108 000000 ------Autumn 21x103 000000 ------Winter 3x109 0 0 0 0 2x108 108 ------Spring05 44x107 16x107 5 x107 9 x107 107 108 ------Annual mean 8 x108 3x107 107 0 18x106 4x107 4x107 El El-Mex Spring 04 3x1010 000000 Drainage ------Summer 29x107 000000 ------Autumn 6x104 000000 ------Winter 24x108 000000 ------Spring 05 4x108 000000 ------Annual mean 7 x108 00000 El-Agami Spring 04 31x106 00106 000 ------Summer 28x106 000000 ------Autumn 5x103 000000 ------Winter 28x108 00000108 ------Spring 05 8x106 8x105 19 x105 13 x105 13 x105 106 7 x105 ------Annual mean 6 x108 16x104 38x104 46x104 26x104 2x105 2x107 a*= S.aureus, b*=S.faecalis, c*=P.aeruginosa, d*= M.luteus, e*= E.coli, f*= C.albicans and 0= no count

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Table 5: Inhibition zone(mm) of the promising isolates against the different pathogens. Isolate Source Antimicrobial Activity Against Pathogenic Strains ------E.coli S. aureus S. faecalis P. aeruginosa M. luteus C. albicans 1 El- Mex Drainage (W) 0 0 12 0 0 0 ------2 El-Mex (S) 0 0 0 0 0 14 ------3 0 13 15 17 0 0 ------4 0 18 0 0 0 17 ------5El-Shatby (S)1416161400 ------6 0000180 ------7 018001615 ------8019016160 ------9 El- Mex Drainage (W) 0 0 0 0 14 11 ------10 0 0 12 0 0 13 ------11 Sidi-Bishr (W) 15 16 0 0 18 0 ------12 Sidi-Bishr (S) 0 20 0 18 22 16 ------13 12 0 0 0 14 0 ------14 24 28 26 23 32 22 ------15 El- Mex D. (S) 0 18 20 0 14 0 ------16 Abu-Qir (S) 0 0 0 0 0 14 ------17 Abu-Qir (W) 0 0 0 0 0 18 ------18 Stanley (W) 0 12 0 0 0 13 ------19 0000180 ------20 El- Mex (W) 0 14 0 0 0 0 ------21 0 15 0 0 0 17 ------22 12 0 0 11 13 12 ------23 El-Montazh (W) 14 12 0 10 12 13 ------24 00001414 ------25 0 0 0 12 17 14 ------26 Sidi-Bishr (S) 10 8 0 10 0 12 ------27 Sidi-Bishr (W) 0 12 0 0 14 0 ------28 Sidi-Bishr (S) 0 16 20 18 20 16 ------29 Qaietbay (S) 22 23 30 24 34 18 ------30 Sidi-Bishr (S) 19 15 0 22 23 16 ------31 20 21 8 12 10 0 ------32 0 0 0 0 0 16 ------33 Abu-Qir (W) 0 0 0 0 0 14 ------34 0 0 0 0 0 10

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3.5.Clustering of bacteria using the SsM strain identified as Flavobacterium sp. (class: coefficient with the UPGMA algorithm: 34 promising flavobacteria). strains were subjected to morphological, physiological and biochemical characterization (data not shown). Phenon b: Members of this phenon were clustered at They were classified into two groups. The first group 93.5% similarity. They were isolated from El-Montazah contained 28 strains and representing the Gram positive sea water and sediments of El-Shatby. The strains. The second group representing the Gram representative strain identified as Moraxella sp. (class: negative strains and contained only 6 strains. Each gamma-protobacteria). One of the two unclustered group was further clustered using the SsM coefficient strains identified as Acinetobacter sp. (class: gamma- with UPGMA algorithm. Results of the numerical study protobacteria). are shown in the dendograms (Fig. 2a,b). The Gram positive strains are grouped into 3 Discussion: The growing interest in marine phena (A, B and C) at 90% similarity level. microbiology is due in part to the existence of species with properties which enable them to survive and grow Phenon A: This minor group harbored only 2 strains under the often unfavourable conditions in the marine which were clustered at 93% similarity. One strain was environment. The role of these bacteria in the sea is isolated from sediments of Sidi-Bishr and the second not known but their special inhibitory properties may from sediments of Kaietbay. play some part in determining the relationships within the microbial ecology of the sea. Many marine Phenon B: This phenon was the intermediate group heterotrophic bacteria are known to produce that harboured 6 strains which clustered at 91% antimicrobial substances which inhibit or kill other similarity. Two of them were isolated from sea water organisms. Isolation and characterization of anti- of Stanley, 3 were from sea water of Abu-Qir, El- microbial agents have been reported by many Montazah and El-Mex. El-Mex sediment included only researchers [20,21] A great percentages of these marine one strain. microorganisms have not been described [22]. The observed physico-chemical characteristics of Phenon C: This phenon is the main group that sea water and sediments in the sites under investigation included 17 strains which clustered at 94% similarity. are similar to those obtained in previous studies [23]. These strains were isolated from different sites. Sea Distribution of antistaphylococcus group in sea water comprised 7 strains represented in El-Mex, El- water was favourable during spring 2005 which is

Mex Drainage and Sidi-Bishr (each site included 2 attributed to nutrient levels (NH4, NO3 and PO4) which strains) while El-Montazah included only one strain. exhibited positive significant correlations with counts of Sediments harboured 10 strains which were distributed this bacterial group (r=0.765, 0.899 and 0.952) in El-Shatby, Sidi-Bishr (included 3 strains), El-Mex (2 respectively. Significant effect of nutrients was also strains) while Abu-Qir and El-Mex Drainage comprised reported by Repka, et al.[24] Salinity exhibited negative one strain for each. significant correlation with counts of the same bacterial One strain of each phenon was selected and group during this season (r=-0.954). The same effect of identified by the aid of Bergy,s Manual of Systematic salinity was reported by Pursell et al.[25] while Lunestad Bacteriology. Member of phenon A was identified as and GoksQy [26] reported its positive significance on the Enterococcus sp. ( class: firmicutes) and member of count. The highest count recorded at El-Montazah phenon B was identified as Arthrobacter sp. (class: during winter which may be due to increasing the actinobacteria) while member of phenon C was activity of bioactive compounds-producing bacteria . identified as Micrococcus sp. (class: actinobacteria). In Absence of bacterial distribution was seen at Abu-Qir addition to the three mentioned phena, the dendogram and El-Mex Drainage due to the unfavourable contained 3 single strains unclustered at 90% similarity conditions prevailed at these sites represented in high level. One strain was identified as Corynebacterium sp. disposal of sewage effluents, agricultural effluents and ( class: actinobacteria) , the second was identified as companies effluents which cause an increase in Bacillus sp. (class: firmicutes) and the third was not temperature and the toxic substances which inhibit the identified. antimicrobial activities. At 94% similarity level, the Gram negative strains We can conclude that, the broad seasonal were clustered into 2 phena at 94% (a,b), each of them distribution of bacterial groups exhibiting antimicrobial harboured 2 strains and there were 2 single unclustered activity in sea water and sediments was for winter and strains. spring seasons. Absence of activity was usually recorded during summer and autumn due to the Phenon a: Members of this phena were clustered at unfavourable and dry conditions prevailed during these 97% similarity. They were isolated from sea water of successive seasons. Abu-Qir and El-Mex Drainage .The representative

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Fig. 2: Simplified dendogram showing the relationships among clusters of Gram positive strains (A) and Gram negative strains (B) based on the Ssµ-UPGMA analysis.

The distribution of bacteria exhibiting antimicrobial Studies examining the frequency of antagonistic activity depends on the location and source of interactions of marine bacterial isolates have found that wastewater. Their marked absence or presence in low 5-8% of the isolates express some level of activity [27]. numbers at some sites such as El-Mex Drainage in The cell-specific levels of activity of particle-attached spite of having high nutrients level suggest that high bacteria are often 2-3 orders of magnitude greater than nutrient levels may not necessitate or inhibit the those of co-occuring, free-living bacteria [28]. In the synthesis of antimicrobial compounds. It is also present study, the percentage of the bio-active possible that these microorganisms are not competing compounds producing bacteria reached up to 30.43% in for the same nutrients or other resources as shown also El-Agami sea water during spring 2005 and up to by Nair and Simidu [27]. 36.36% at El-Mex sediments during the same season. Competition amongst microbes for space and Thirty four bacterial isolates were selected for nutrients in the marine environment is a power antagonistic studies against six identified pathogens. It selective force which has led to the evolution of a was found that the majority of these microorganisms variety of effective strategies for colonizing and were Gram positive (84%) while only (16%) growing on surfaces. Long and Azam [6] hypothesized represented the Gram negative group. This finding was that bacterium-bacterium antagonistic interactions may reported in other studies [29]. Nonetheless, later studies contribute to variations in community structure at confirmed that Gram-negative producers also occur [30]. microscale. Furthermore, the species richness and Enterococcus species of phenon A are known to diversity on particles could be influenced by bacterium- produce bioactive compounds including bacteriocins bacterium antagonism, and in turn, this could affect the and antibiotics against different types of pathogens nature and rates of biogeochemical transformation of [20,31], in addition to using them as probiotics in the particles. aquacultures to inhibit fish pathogens [32]. The

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representative strain of phenon B was identified as such as vaccination and use of antimicrobial agents are Arthrobacter sp. This genus inhabiting the marine employed in aquaculture as in other areas of animal environment as reported by Beleneva and Zhukova [33]. production. Production of bioactive compounds by this species was also reported [34]. The representative member of phenon Conclusion: marine isolates from Alexandria coasts are C was identified as Micrococcus sp. Micrococcus sp. valuable source for bioactive compounds production, belongs to family Micrococcaci. Genetic studies have which can be used as probiotics in aquaculture or in suggested that Micrococcus belongs to the actinomycete pharmaceutical applications. group, and the isolates originally identified as species of Micrococcus have since been placed in five different REFERENCES genera [35]. Previously, both Staphylococci and Micrococci were included in the same genus 1. Wagner-Dobler, I.,W. Beil, M. Meiners and H. Micrococcus. Most of Micrococcus spp. lives on skin Laatsch, 2002. Integrated approach to explore the and other external surfaces of animals [36]. Micrococcus potential of marine microorganisms for the sp. were also isolated from different marine sources [34] production of bioactive metabolites. Adv. Biochem. and exhibiting different antimicrobial activities [37]. Eng. Biotechnol., 74: 207-238. Detection of Bacillus sp. in marine environment was 2. Blunt, J.W., B.R. Copp, M.H.G. Munro, P.T. reported, applications of its secondary metabolites as Northcote and M.R. Prinsep, 2004. Marine natural antimicrobial agents and probiotics have been discussed products. Nat. Prod. Rep., 21: 1-49. [38]. Corynebacterium sp is a Gram positive, 3. Anand, T.P., A. W. Bhat, Y.S. Shouche, U. Roy, nonpathogenic bacterium. Certain products and by- J. Siddharth and S.P. Sharma, 2006. Antimicrobial products of naturally-occurring metabolic processes in activity of marine bacteria associated with sponges cells have utility in a wide array of industries, from the waters off the coast of South East. India. including the food, feed, cosmetics, and pharmaceutical Microbiol. Res., 161: 252-62. industries. These molecules, collectively termed fine 4. Uzair, B., N. Ahmed, V.U. Ahmad, F.V. chemicals, include organic acids, both proteinogenic Mohammad and D.H. Edwards, 2008. The and non-proteinogenic amino acids, nucleotides and isolation, purification and biological activity of a nucleosides, lipids and fatty acids, diols, carbohydrates, novel antibacterial compound produced by aromatic compounds, vitamins and cofactors, and Pseudomonas stutzeri. FEMS Microbiol. Lett., 279: enzymes [39]. Flavobacterium sp. was representative for 243-240. phenon a, The class Flavobacteria can dominate many 5. Burgess, J.G., H. Miyashita, H. Sudo, and T. marine systems and are often associated with marine Matsunaga,1991. Antibiotic production by the snow and the decay of algal blooms [40]. They were marine photosynthetic bacterium Chromatium found throughout the water column of marine purpuratum NKPB 031704: Localization of activity environment [41]. Glöckner et al.[42] reported that the to the chromatophores. FEMS Microbiol. Lett., 84: class flavobacteria were the most abundant group 301-306. detected in a number of different marine systems, 6. Long, R.A. and F. Azam, 2001. Antagonistic accounting for (2 to 72 %) of the stained cells. interactions among marine pelagic bacteria", Appl. Production of bioactive compounds was reported Environ. Microbiol., 67(11): 4975-4983. by [43]. The selected strain of phenon b was identified 7. International Organization for Standardization as Moraxella sp..The existence of this species in (ISO 5667/2), 1990. Water Quality- sampling- part marine environment was reported [34]. Acinetobacter sp. 2: Guidance on sampling techniques. Geneva, has been isolated from sea water, sediments as well as Switzerland, pp: 9. hypesaline areas. It has been reported as a free living 8. American Public Health Association (APHA), bacterium in marine water and sediment. It is also 1999. Standard Methods for the Examination of found attached to different substrates such as sea grass, Water and Wastewater, 19th ed. Clesceri, L.S; algae or other material. It contributes to the normal Greenberg , A.E. and Eaton, A.D.(eds), American flora of marine macro organisms like fishes, crabs, Public Health Association, American Water Works prawns etc. Production of novel metabolites by this Association, and Water Environment Federation, marine Acinetobacter sp. is one of the important Washington, D.C. aspects [44]. 9 Intergovernmental Oceanographic Commission Aquaculture is growing rapidly in many regions of (IOC), 1983. Chemical methods for use in marine the world, and aquaculture products constitute an environmental monitoring manual and important food supply with increasing economic guides.UNESCO, pp: 53. importance. To control infectious diseases, strategies

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