Isolation of marine bacteria, antagonistic to human pathogens

Transcription

1 Indian Journal of Marine Sciences Vol. 31(1), March 2002, pp Isolation of marine bacteria, antagonistic to human pathogens K. Jayanth, G. Jeyasekaran* & R. Jeya Shakila Department of Fish Processing Technology, Fisheries College & Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Tuticorin , TN, India Received 3 April 2001; revised 27 August 2001 Pigmented bacteria from marine samples including seawater, sediment, seaplants, bivalves and submerged substrates of the Tuticorin coast were screened for the antibacterial activity. Of the 162 pigmented bacteria, 62 exhibited antagonism towards the indicator organisms Lactobacillus, Arthrobacter and Micrococcus. Alteromonas was the dominant antagonistic marine bacteria, exhibiting a wide antibacterial spectrum against the human pathogens associated with seafoods such as Staphylococcus aureus, Salmonella typhi and Vibrio cholerae. Results indicated that antibacterial substances present in the antagonistic marine bacteria could be used to inhibit the growth of human pathogens. [ Key words : Bacteria, pathogens, antagonism, seafoods, human pathogens ] Ocean is a promising source of novel bioactive compounds. Macroorganisms like holothurians, sponges, etc. have proven to be good sources although their isolation, purification and large scale production have proven to be difficult. On the other hand, marine microorganisms, which produce bioactive substances 1, could serve as an alternative source, as it is comparatively easier to isolate and purify them. Marine bacteria are known to produce inhibitory substances in the marine environment, even if they are not specifically antibiotic producers. Marine bacteria showing antibacterial activities have been isolated from various biotopes such as surface or deep sediments, seaweeds and other substrates. The bacteria so far identified mostly belong to the species of Bacillus, Micrococcus, Pseudomonas, Vibrio, Flavobacterium, Alcaligenes, Xanthomonas and Achromobacter 1-3. Some of these antagonistic bacteria show antibacterial activity against bacterial pathogens. The antibacterial substances produced by these antagonistic bacteria could be further extracted, characterized and used as antibacterial substances to control the growth of human pathogens associated with seafoods. Hence, the present investigation was undertaken to isolate the antagonistic bacteria present in the marine environment of Tuticorin coast of Tamil Nadu and to examine their inhibitory action against selected human pathogens associated with seafoods. *Corresponding author E.mail : jerosh@vsnl.com Materials and Methods A total of 101 samples from seawater, sediment, seaplants (Gracilaria, Zostrea), bivalves (Donax, Paphea) and swabs from the submerged substrates such as rock, boat, wooden poles and oyster shells were collected from five different stations viz. Therespuram, Fishing Harbour, Roche Park, Thermal Beach and Hare Island of Tuticorin coast of Tamil Nadu. All the samples were brought to the laboratory within an hour of collection. Bacteria were isolated from these samples after making serial dilution using sterile half strength (HS) seawater. The total culturable bacterial and pigmented bacterial counts were determined on seawater yeast extract peptone (SYEP) medium following spread plate technique 4. The seawater yeast extract contains peptone (5 g w/v), yeast extract (3 g w/v), aged seawater (750 ml) and distilled water (250 ml) with a ph of 8.0. Pigmented colonies were picked at random from each plate and streaked onto SYEP agar to obtain pure cultures and then used to test antagonistic activity. Indicator organisms were selected from the predominant marine bacteria isolated from the marine environment, based on their sensitivity to antibiotics for the screening of antagonistic marine bacteria. Antibiograms were made by agar disc diffusion assay on Mueller Hinton agar supplemented with 1.5% (w/v) sodium chloride 5. The bacteria showing larger zones of inhibition were selected as indicator organisms and they were identified up to generic level following the standard taxonomic scheme 6. Cross streak method

2 40 Indian J. Mar. Sci., Vol. 31, No. 1, March 2002 was used for assaying the inhibitory activity of pigmented bacteria 7. Fresh cultures (20 h old) were streaked (4-6 mm width) across the diameter of SYEP agar (1.2% agar) plates and cultures of indicator bacteria were streaked at right angles across them. Plates were incubated at 30 ± 2 C for another 24 h. The bacteria which inhibited the growth of the indicator organism in the confluence area are termed as antagonistic bacteria; and were identified up to generic level 6. Double agar overlay method was used for the assay of antagonistic bacteria against the human pathogens associated with seafoods 8. The human pathogens, which were used as test organisms in this study, were Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, S. typhi and Vibrio cholerae. Macro-colonies of antagonistic marine bacteria were developed on SYEP agar (1.2% agar) plates by inoculating 18 h old SYEP broth culture with a micropipette and incubated at 30±2 C for 40 h. The colonies grown were killed by exposure to the chloroform vapour for min. All the test organisms were grown in Trypticase soy medium and incubated at 37 C for 18 h. About 10 μl of the culture was suspended in 8 ml of soft SYEP agar (0.7% agar) maintained at C and poured immediately over the macro-colonies of the antagonistic marine bacteria on the SYEP agar. The plates were incubated for 24 h at 37 C. The cleared zone around the macrocolony of the antagonistic marine bacteria was measured. A control plate without macro-colonies of the antagonistic marine bacteria was used to examine the possible effect of chloroform on the growth of test organisms. Two strains of Alteromonas, which showed higher antagonistic activity, were selected for the extraction of antibacterial substance produced by them using the solvent ethyl acetate. These strains were inoculated onto SYEP agar (1.2% agar) plates to give thick, profuse growth and incubated for 48 h at 30 ± 2 C. The cells were then gently scrapped and washed off with HS seawater. The suspension was centrifuged at 10,000 rpm at 4 C for 15 min. The cell pellet was suspended in ethyl acetate to extract the antibacterial substance 9. The ethyl acetate extract was evaporated at C to give a concentrated crude extract of the antibacterial substance. The spent agar medium was also sectioned, homogenized with ethyl acetate and allowed to stand for 3 h without agitation at 30 ± 2 C. The solvent decanted and evaporated at C to give a crude extract of the antibacterial substance. Simple correlation and two-way analysis of variance technique were used to test the significance by following the standard procedure 10. Results and Discussion Pigmented marine bacteria were enumerated for the isolation of antagonistic marine bacteria. From 101 marine samples, 166 pigmented bacteria were isolated. The pigmented bacterial population was lower by about 2-3 log counts than the total culturable bacterial population (Fig. 1). The proportion of pigmented bacteria in different samples of the marine environment showed wide variance, ranging from < 0.19% to 45.55%; < 0.10% to 33.33%; < 1.27% to 11.10%; 3.13% to 20.15%; 0.07% to 38.07% in seawater, sediment, seaweeds, bivalves and submerged substrates, respectively. Pigmented marine bacteria were selected and tested for antagonistic activity, because earlier studies have demonstrated that most of the antibiotics producing marine bacteria were pigmented 7. A significant positive correlation (P < 0.5) existed among the pigmented and total culturable bacterial populations. In another study 11, the proportion of pigmented bacteria was found to be 30%. However, in the present study, the proportion of pigmented bacteria varied with different samples and out of the total number of samples analyzed, it was only 6%. Fig. 1 Comparison of pigmented bacterial counts with the total culturable bacterial load in different marine samples (A. Seawater; B. Sediment; C. Seaplants; D. Bivalves; E. Submerged substrates)

3 Jayanth et al..: Isolation of marine bacteria 41 The indicator organisms were selected from the predominant bacteria isolated from the marine environment and were identified as Gram-positive Micrococcus (SI), Lactobacillus (S2) and Arthrobacter (S3). It is relevant to note that Gram-positive bacteria are more susceptible than Gram-negative bacteria to the antibiotics derived from the marine microorganisms 12. Of the 166 pigmented bacteria isolated from the marine samples, 62 bacteria (37.35%) were found to show antagonism against the indicator organisms (Table 1). The proportion of antagonistic marine bacteria was found to be high in seaplants (35.48%), followed by bivalves (30.63%). It has been reported that the occurrence of antagonistic marine bacteria was high in seaplants, as there exists a beneficial relationship between the antagonistic bacteria and seaweeds/algae 7 and animals that harbour them 3. However, the occurrence of bacteria to the total culturable bacterial population among the different marine samples did not show any significant correlation (P < 0.05). The proportion of antagonistic marine bacteria in the samples collected from different stations was also quite variable (Table 1). Presence of antagonistic marine bacteria was high in Fishing Harbour, followed by Therespuram, Roche Park, Hare Island and Thermal Beach. Higher incidence of antagonistic bacteria in the Fishing Harbour may be because of the favourable condition that exists in the area. It has been earlier reported that constant nutritional inputs from external sources assuage the necessity for bacterial populations to produce antibacterial compounds to survive competition 11. However, in this study, the incidence of antagonistic marine bacteria did not show any significant relationship (P< 0.05) with the different sampling stations. It was observed that majority of the antagonistic bacteria were Gram-negative and only one strain was Gram-positive. The bacteria that exhibited antagonistic activity were Flavobacterium, Alteromonas, Pseudomonas/ Alteromonas/ Flavobacterium group and Micrococcus. The proportion of Flavobacterium was high (67.74 %), followed by Alteromonas (29.03 %) and other bacteria. Flavobacterium was found in all the samples of seaweeds and bivalves. The antagonistic bacteria isolated from the marine environment exhibited variable inhibitory activity against the indica- Table 1 Proportion of antagonistic marine bacteria with reference to different marine samples and different sampling stations Source Antagonistic bacteria No. of antagonistic bacteria I II III IV I. Marine samples Seaweeds 9 (14.52)* Sediment 3 (4.84) 3 Seaplants 22 (35.48) 15 7 (31.82) Bivalves 19 (30.64) 8 11(57.89) Submerged 9 (14.52) 9 Substrates II. Sampling stations Therespuram 19 (30.65) 14 (73.68) 5 (6.32) Fishing 26 (41.95) 18 8 (30.77) (69.23) Harbour Roche Park 8 (12.90) 4 (50.00) 4 (50.00) Thermal 2 (3.25) 1 (50.00) 1 (50.00) Beach Hare Island 7 (11.29) 5 (71.43) 1 (14.29) 1 (14.29) Total 62 (100.00) 42 (67.74) 18 (29.03) 1 (1.61) 1 (1.61) *Percentage values are given in parentheses I Flavobacterium; II Alteromonas; III Pseudomonas/ Flavobacterium; IV - Micrococcus

4 42 Indian J. Mar. Sci., Vol. 31, No. 1, March 2002 tor organisms. Alteromonas was found to exhibit a wide spectrum of antibacterial activity, although Flavobacterium was recorded in highest numbers. Several workers have also recorded higher incidence of Flavobacterium and Alteromonas and their inhibitory activity against many bacteria 1,2,11. Antagonistic marine bacteria, which were tested for their ability to inhibit the growth of test organisms, showed that out of the 62 antagonistic marine bacteria, 18 (29.03 %) had the ability to inhibit at least any one of the test organisms (Table 2). Alteromonas strains isolated from seaplants and bivalves exhibited antagonism towards all the test organisms, while Flavobacterium showed inhibition against only one test organisms (Staphylococcus aureus). However, the Table 2 Antibacterial spectrum of antagonistic marine bacteria against test organisms by double agar overlay method Source Seaplants Zone of Inhibition (mm) Test organisms T1 T2 T3 T4 T5 T6 F1 x ++ x x x x F2 x ++ x x x x Submerged substrates F3 x ++ x x x x Seaplants A x A x A x A x A x Bivalves A x A x A8 + + x A9 + + x A x A x A x A x A x A x x = 0 mm; + = >1 to 10 mm; ++ = 10 to 20 mm; +++ = 20 to 30 mm; ++++ = > 30mm; F Flavobacterium; A- Alteromonas T1 Listeria monocytogenes NCTC 11994; T2 Staphylococcus aureus ATCC 9144; T3 - Escherichia coli; T4 Salmonella typhimurium ATCC ; T5 S. typhi ATCC 6539; T6 - Vibrio cholerae Pseudomonas/ Alteromonas/ Flavobacterium group and Micrococcus did not show any antagonism towards any of the test organisms. Among the 18 isolates, which showed antagonistic effect, 3 isolates (F1-F3) belonged to the genus Flavobacterium and remaining isolates belonged to the genus Alteromonas (A1-A15). The test organism, E. coli was not inhibited by any of the antagonistic bacteria. The other test orgainsms viz. Listeria monocytogenes, Salmonella typhimurium, were inhibited by all strains of Alteromonas at varied levels. The inhibitory activity of the Alteromonas strains was more pronounced against Staphylococcus aureus, S. typhi and Vibrio cholerae. Among the Alteromonas strains, two strains (A6 and A7) were found to exhibit a wide and marked spectrum of antibacterial activity against the test organisms when compared to other strains. The antagonistic effect of Alteromonas strains against the Staphylococcus aureus, S. typhi and Vibrio cholerae, the potential human pathogens associated with seafoods was very well pronounced than the other pathogens, Listeria monocytogenes and Salmonella typhimurium. Several workers have earlier reported the importance of marine pigmented Alteromonas and it had showed stronger inhibitory activity against various bacterial pathogens, viz. S. aureus, S. epidermidis and Bacillus cereus 7,11,13. The Alteromonas strain isolated in another study 14 also had stronger inhibitory activity against the V. cholerae and S. typhi, as observed in the present study.

5 Jayanth et al..: Isolation of marine bacteria 43 Table 4 Inhibitor activity of ethyl acetate crude extract of Alteromonas strains A6 and A7, against test organisms Zone of Inhibition (mm) Volume Alteromonas strain A6 Alteromonas strain A7 (μl) T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 Crude extract from cell x x x x x x x x x x x x x x x x x x x x x x x x x ++ x x x x x ++ x x x x x x x x x x x x x x x x (Control) Crude extract from spent medium x x x x x x x x x x x x x x x x x x x x x x x x x ++ x x x x x ++ x x x x x x x x x x x x x x x x (Control) x = 0 mm; + = > 1 to 10 mm; ++ = > 10 to 20 mm; +++ = > 20 to 30 mm Control Ethyl acetate T1 Listeria monocytogenes NCTC 11994; T2 Staphylococcus aureus ATCC 9144; T3 - Escherichia coli; T4 Salmonella typhimurium ATCC 23564; T5 S. typhi ATCC 6539; T6 - Vibrio cholerae. Organic solvents have been employed to extract the antibacterial substances produced by the marine bacteria. Among the organic solvents, ethyl acetate has been widely employed 9,15,16. Crude extracts of the antibacterial substance prepared from the two Alteromonas strains cell pellet and spent media were tested against the indicator organisms and the results are presented in Table 3. The crude extract recovered from the cell pellet of Alteromonas strain A6 exhibited inhibitory activity against the indicator organisms S1, S2 and S3 at the levels 10, 50 and 50 μl, respectively, while the crude extract recovered from the Alteromonas strain A7 exhibited inhibitory activity against only two indicator organism S1 and S3 at the levels of 40 and 50 μl, respectively. The crude extract obtained from the spent medium of Alteromonas strain A6 exhibited inhibitory activity against only one indicator organism, S3 at the level of 100 μl. Ethyl acetate, used as control at 100 μl level did not inhibit any of the indicator organisms. The crude extract of the antibacterial substances recovered from the cells of two Alteromonas strains and the spent media were tested for their inhibitory activity against the test organisms and the results are presented in Table 4. The crude extract recovered from cell pellet and spent media of Alteromonas strain A6 and A7 inhibited the growth of Staphylococcus aureus at the same level (200 μl). The results indicate Table 3 Inhibitory activity of ethyl acetate crude extract of Alteromonas strains A6 and A7, against indicator organisms Zone of Inhibition (mm) Alteromonas strain A6 Alteromonas strain A7 Volume S1 S2 S3 S1 S2 S3 (μl) Crude extract from cell 2.5 x x x x x x 5.0 x x x x x x x x x x x x x x x x x x x x x x x + x x x x x 50.0 (Control) x x x x x x Crude extract from spent medium 2.5 x x x x x x 5.0 x x x x x x 10.0 x x x x x x 20.0 x x x x x x 30.0 x x x x x x 40.0 x x x x x x 50.0 x x + x x x 50.0 (Control) x x x x x x x = 0 mm; + = > 1 to 10 mm; ++ = > 10 to 20 mm S1 Micrococcus; S2 - Lactobacillus; S3 Arthrobacter Control Ethyl acetate

6 44 Indian J. Mar. Sci., Vol. 31, No. 1, March 2002 that the antibacterial substance may be closely bound with the cell and secreted into the solid media. It has also been suggested that inhibitory compounds produced by marine bacteria are closely associated with the cell 17. It has been observed that antibiotic produced by P. bromoutilis was found to be better on solid media 15. The antibacterial substances recovered from marine bacteria using ethyl acetate have turned out to be a quinolinol 9, or pyrrole 15 or a polysaccharide 16 and hence the antibacterial compound of the Alteromonas strains may be an organic compound or a polysaccharide. The antibacterial substances present in the antagonistic marine bacteria could be used to inhibit the growth of human pathogens. Further study is required to examine the exact nature of the antibacterial components. Acknowledgement The authors thank the Tamil Nadu State Council for Science and Technology, Govt. of Tamil Nadu for extending financial support to carry out this study. 10 Snedecor G W & Cochran W G, Statistical methods, (Oxford and IBH Publishing Company, Calcutta) 1962, pp Nair S & Simidu U, Distribution and significance of heterotrophic marine bacteria with antibacterial activity, Appl Environ Microbiol, 53 (1987) Gauthier M J & Flatau G N, Antibacterial activity of marine violet pigmented Alteromonas with special reference to the production of brominated compounds, J Microbiol, 22 (1976) McCarthy S A, Johnson R M & Kakimoto D, Characterization of an antibiotic produced by Alteromonas luteoviolacea Gauthier, isolated from Kinko Bay, Japan, J Appl Bacteriol, 77 (1994) Barja J L, Lemos M L & Toranzo A E, Purification and characterization of an antibacterial substance produced by a marine Alteromonas sp., Antimicrob Agents Chem, 33 (1989) Burkholder P R, Pfister R M & Leitz F H, Production of a pyrrole antibiotic by a marine bacterium, Appl Microbiol, 14 (1966) Anderson R J, Wolfe M S & Faulkner D J, Autotoxic antibiotic production by a marine chromobacterium, Mar Biol, 27 (1974) Rosenfeld W D & Zobell C E, Antibiotic production by marine microorganisms, J Bacteriol, 54 (1947) References 1 Bernan V S, Greenstein M & Maisese W M, Marine microorganisms as a source of new natural products, Adv Appl Microbiol, 43 (1997) Gauthier M J, Shewan J M, Gibson D M & Lee J V, Taxonomic position and seasonal variations in marine neritic environment of some Gram-negative antibiotic producing bacteria, J Gen Microbiol, 87 (1975) Austin B,. Novel pharmaceutical compounds from marine bacteria, J Appl Bacteriol, 67 (1989) Schneider J & Rheinheimer G, Isolation methods, in Methods in aquatic bacteriology, edited by B Austin, ( John Wiley & Sons Ltd., New York) 1988, pp Bauer AW, Kirby W M M, Sherris J S & Turk M, Antibiotic susceptibility testing by a standardised single disc method, American J Clinical Pathol, 45 (1966) LeChavelier M W, Seider R J & Evans E M, Enumeration and characterization of standard plate count bacteria in chlorinated and raw water supplies, Appl Environ Microbiol, 40 (1980) Lemos M L, Toranzo A E & Barja J L, Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds, Microbial Ecol, 11 (1985) Dopazo C P, Lemos M L, Lodeiros C, Bolinches J Barja J L & Toranzo A E, Inhibitory activity of antibiotic producing marine bacteria against the fish pathogens, J Appl Bacteriol, 65 (1988) Wratten S J, Wolfe M S, Andersen R J & Faulkner D J, Antibiotic metabolites from the marine Pseudomonad, Antimicrob Agents Chem, 11 (1977)