Sains Malaysiana 43(4)(2014): 543–550

Population Density and Antibiotic Resistant of Bacteria from Bivalve

(Perna viridis and Anadara granosa)

(Kepadatan Populasi dan Kerintangan Antibiotik oleh Bakteria daripada Bivalvia

(Perna viridis dan Anadara granosa))

ASMAT AHMAD¹*, NUR DIANA MEHAT², RAHIMI HAMID¹& GIRES USUP²

¹School of Biosciences and Biotechnology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

²School of Environmental and Natural Resource Sciences, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

Diserahkan: 19 Februari 2013/Diterima: 5 Ogos 2013

ABSTRACT

This study was carried out to know the bacteria population density in the blood cockle (Anadara granosa) and green lipped mussel (Perna viridis), to analyse the bacteria resistance towards antibiotics and antimicrobial activity of isolates against selected pathogen. Samples of blood cockle and green lipped mussel were obtained from five areas in Kedah and Negeri Sembilan. Bacterial population densities in mussels and cockles were 3 × 10² - 8 × 10⁸ CFU/mL and 5 × 10² - 5 × 10⁸ CFU/mL, respectively. A total of 162 isolates were obtained, of which 131 isolates were from mussels and 31 isolates were from cockles. Vibrio sp. was the most dominant genus in both types of samples. Antibiotic testing of all isolates showed most were resistant to Penicillin (10 U) and most were sensitive to Ciprofloxacin (5 µg). Most isolates (160/162) showed resistance to at least two antibiotics and 10 isolates were resistant to more than five antibiotics. Multiple antibiotic resistance indices (MAR) were calculated based on the antibiotic resistance results. Most isolates had a MAR index value of 0.2 which indicated the isolates were not contaminated with antibiotic residues. The highest index value was 0.7. Fifteen out of 39 isolates which produced beta-lactamase enzyme were tested for antimicrobial activity against selected pathogen. Results indicated that antimicrobial activity were varies among the isolates. Isolate SMII-Ip produced antimicrobial activity against six out of the nine tested pathogen and none of the isolates active against Pseudomonas mirabilis.

Keywords: Anadara granosa; antibiotic; antimicrobial; population density; Perna viridis

ABSTRAK

Kajian ini dijalankan bagi mengetahui kepadatan populasi bakteria daripada kerang (Anadara granosa) serta kupang (Perna viridis), menganalisis kerintangan bakteria terhadap antibiotik serta aktiviti antimikrob oleh pencilan terhadap patogen pilihan. Sampel kerang dan kupang telah diperolehi dari lima kawasan perairan Kedah dan Negeri Sembilan. Kepadatan populasi bakteria pada kupang adalah 3 × 10² - 8 × 10⁸ CFU/mL dan kerang 5 × 10² - 5 × 10⁸ CFU/mL Sebanyak 162 pencilan telah berjaya dipencilkan, dengan 131 pencilan adalah daripada kupang dan 31 daripada kerang. Vibrio sp. merupakan genus paling dominan daripada kedua-dua sampel. Ujian kerintangan antibiotik terhadap semua pencilan menunjukkan kebanyakan isolat rintang terhadap Penisilin (10 U) dan sensitif terhadap Ciprofloksasin (5 µg). Hampir semua pencilan (160/162) rintang terhadap sekurangnya-kurangnya dua antibiotik dan 10 pencilan rintang terhadap lebih daripada lima antibiotik. Kiraan indeks Antibiotik Pelbagai Rintang (MAR) berdasarkan hasil ujian kerintangan antibiotik telah dijalankan. Kebanyakan isolat mempunyai nilai indeks MAR 0.2 yang bermakna pencilan tidak terdedah kepada pencemaran antibiotik. Nilai indeks MAR tertinggi adalah 0.7. Sebanyak 15 daripada 37 pencilan yang menghasilkan enzim beta-laktamase telah diuji aktiviti antimikrob terhadap mikrob pathogen terpilih. Hasil menunjukkan aktiviti antimikrob yang berbeza bagi pencilan yang berbeza. Pencilan SMII-Ip menghasilkan aktiviti antimikrob terhadap enam daripada sembilan pathogen yang diuji dan tidak terdapat pencilan yang aktif merencat Pseudomonas mirabilis.

Kata kunci: Anadara granosa; antibiotik; antimikrob; kepadatan populasi; Perna viridis

RUJUKAN

Bansemir, A., Blume, M., Schröder, S. & Lindequist, U. 2006. Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria. Aquaculture 252: 79-84.

Bauer, A.W., Kirby, W.M.M., Sherris, J.C. & Turck, M. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45: 493-496.

Braithwaite, R. & McEvoy, L. 2005. Marine biofouling on fish farms and its remediation. Adv. Mar. Biol. 47: 215-252.

Brandi, G., Sisti, M., Giardini, F., Schiavano, G.F. & Albano, A. 1999. Survival ability of cytotoxic strains of motile Aeromonas spp. in different types of water. Lett. Appl. Microbiol. 29: 211-215.

Burkhardt, W. & Calci, K.R. 2000. Selective accumulation may account for shellfish associated viral illness. Applied and Environmental Microbiology 66(4): 1375-1378.

Cai, J., Li, J., Thompson, K.D., Li, C. & Han, H. 2007. Isolation and characterization of pathogenic of Vibrio parahaemolyticus from diseased post-larvae of abalone Haliotis diversicolor suprasexta. J. Basic Microbiol. 47: 84-86.

Castro, D., Pujalte, M.J., Lopez-Cortes, L., Garay, E. & Borrego, J.J. 2002. Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): Numerical taxonomy and antibacterial activities. Journal of Applied Microbiology 93: 438-447.

Cavallo, R.A., Acquaviva, M.I. & Stabili, L. 2009. Culturable heterotrophic bacteria in seawater and Mytilus galloprovincialis from a Mediterranean area (Northern Ionian Sea-Italy). Environ. Monit. Assess. 149(1-4): 465-475.

Chitanand, M.P., Kadam, T.A., Gyananath, G., Totewad, N.D. & Balhal, D.K. 2010. Multiple antibiotic resistance indexing of coliforms to identify high risk contamination sites in aquatic environment. Indian J. Microbiol. 50: 216-220.

Defossez, J.M. & Hawkins, A.J.S. 1997. Selective feeding in shellfish: Size dependent rejection of large particles within pseudofaeces from Mytilus edulis, Ruditapes philippinarum and Tapes decussatus. Marine Biology 129(1): 139-147.

Dunphy, B.J., Hall, J.A., Jeffs, A.G. & Wells, R.M.G. 2006. Selective particle feeding by the Chilean oyster, Ostrea chilensis: Implications for nursery culture and broodstock conditioning. Aquaculture 261(2): 594-602.

Elhadi, N., Radu, S., Chen, C.H. & Nishibuchi, M. 2004. Prevalence of potentially pathogenic Vibrio species in the seafood marketed in Malaysia. Journal of Food Protection 67(7): 1469-1477.

Gueguen, Y., Herpin, A., Aumelas, A., Garnier, J., Fievet, J., Escoubas, J.M., Bulet, P., Gonzalez, M., Lelong, C., Favrel, P. & Bachère, E. 2006. Characterization of a defensing from the oyster Crassostrea gigas: Recombinant production, folding, solution structure, antimicrobial activities, and gene expression. J. Biol. Chem. 281: 313-323.

Heidelberg, J.F., Heidelberg, K.B. & Colwell, R.R. 2002. Bacteria of the γ-subclass Proteobacteria associated with zooplankton in Chesapeake Bay. Applied and Environmental Microbiology 68: 5498-5507.

Holmes, P., Niccolls, L.M. & Sartory, D.P. 1996. The ecology of mesophilic Aeromonas in the aquatic environment. Applied Microbiology 17: 58-60.

Huang, C.H., Renew, J.E., Smeby, K.L., Pinkerston, K. & Sedlak, D.L. 2001. Assessment of potential antibiotic contaminants in water and preliminary occurrence analysis. Water Resour. Update 120: 30-40.

Huss, H. 1997. Control of indigenous pathogenic bacteria in seafood. Food Control 8: 91-98.

Kueh, C.S. & Chan, K.Y. 1985. Bacteria in bivalve shellfish with special reference to the oyster. J. Appl. Bacteriol. 59(1): 41-47.

Kümmerer, K. 2009. Antibiotics in the aquatic environment: A review-Part II. Chemosphere 75: 435-441.

Lee, J.K., Jung, D.W., Eom, S.Y., Oh, S.W., Kim, Y.J., Kwak, H.S. & Kim, Y.H. 2008. Occurrence of Vibrio parahaemolyticus in oysters from Korean retail outlets. Food Control 19: 990-994.

Lees, D. 2000. Viruses and bivalve shellfish. Int. J. Food Microbiol 59: 81-116.

Lynn, M. & Solotorovsky, M. 1981. Chemotherapeutic Agents for Bacterial Infections. Stroudsburg: Hutchison Ross Publishers.

Maktoob, A. & Ronald, H.T. 1997. Handbook of Natural Products from Marine Ivertebrates. Phyllum mollusca Part. 1. Harwood: Academic Publishers.

Martinez, J.L. 2003. Recent advances on antibiotic resistance genes. In Recent Advances in Marine Biotechnology: Molecular Genetics of Marine Organisms, edited by Fingerman, N. New Hampshire: Science Publishers. pp. 13-32.

Mazel, D. & Davies, J. 1999. Antibiotic resistance in microbes. Cellular and Molecular Life Sciences 56: 742-754.

Medeiros, A.A. 1997. Evolution and dissemination on β-Lactamase accelerated by generations of β-lactam antibiotics. Clinical Infection Disease 24: 519-545.

Nonaka, L., Isshiki, T. & Suzuki, S. 2000. The occurrence of oxytetracycline-resistant bacteria in the fish intestine and the seawater environment. Microbes. Environ. 15: 223-228.

Olafsen, J.A., Mikkelsen, H.V., Giaver, H.M. & Hansen, G.H. 1993. Indigenous bacteria in hemolymph and tissues of marine bivalves at low temperatures. Appl. Environ. Microbial. 59: 1848-1854.

Olicard, C., Renault, T., Torhy, C., Benmansour, A. & Bourgougnon, N. 2005. Putative antiviral activity in hemolymph from adult Pacific oysters, Crassostrea gigas. Antiviral Res. 66: 147-152.

Oliver, J.D. 1989. Foodborne Bacterial Pathogens. New York: Marcel Dekker Inc.

Pinera-Pasquino, L. 2006. Patterns of antibiotic resistance in bacteria isolated from marine turtles. Master Thesis. College of Charleston, Charleston, South Carolina (Unpublished).

Prieur, D., Mevel, G., Nicolas, J.L., Plusquellec, A. & Vigneulle, M. 1990. Interactions between bivalve molluscs and bacteria in the marine environment. Oceanography and Marine Biology Annual Review 28: 277-352.

Projan, S.J. & Bradford, P.A. 2007. Late stage antibacterial drugs in the clinical pipeline. Curr. Opin. Microbiol. 10: 441-446.

Pujalte, M.J., Ortigosa, M., Macian, M.C. & Garay, E. 1999. The annual cycle of aerobic and facultative anaerobic marine bacteria associated with Mediterranean oysters and seawater. International Microbiology 2: 259-266.

Roch, P., Yang, Y., Toubiana, M. & Aumelas, A. 2008. NMR structure of mussel mytilin, and antiviral–antibacterial activities of derived synthetic peptides. Dev. Comp. Immunol. 32: 227-238.

Ronald, J.A., Breena, M. & Melissa, M. 2002. Antibiotic resistance of Gram negative bacteria in Rivers, United States. Emerging Infectious Disease 8(7): 1-9.

Salyers, A.A., Gupta, A. & Wang, Y. 2004. Human intestinal bacteria as reservoirs for antibiotic resistance genes. Trends Microbiol. 12: 412-416.

Santos, O.C.S., Pontes, P.V.M.L., Santos, J.F.M., Muricy, G., Giambiagi-deMarval, M. & Laport, M.S. 2010. Isolation, characterization and phylogeny of sponge associated bacteria with antimicrobial activities from Brazil. Research in Microbiology 161: 604-612.

Sarter, S., Nguyen, H.N.K., Hung, L.T., Lazard, J. & Montent, D. 2007. Antibiotic resistance in Gram negative bacteria isolated from farmed catfish. Food Control 18: 1391-1396.

Smith, J.J., Howington, J.P. & McFeters, G.A. 1993. Plasmid maintenance and expression in Escherichia coli exposed to the Antarctic marine environment. Antarctic Journal of the United States 28: 123-124.

Suantika, G., Dhert, P., Rombaut, G., Vandenberghe, J., De Wolf, T. & Sorgeloos, P. 2001. The use of ozone in a high density recirculation system for rotifers. Aquaculture 201: 35-49.

Thavasi, R., Apernavedi, S., Jayalakshimi, S. & Balasubramanian, T. 2007. Plasmid mediated antibiotic resistance in marine bacteria. Journal of Environmental Biology 28(3): 617-621.

Thompson, F.L., Iida, T. & Swings, J. 2004. Biodiversity of Vibrios. Microbiology and Molecular Biology Reviews 68: 403-431.

Urakawa, H., Yoshida, T., Nishimura, M. & Ohwada, K. 2000. Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling. Environ. Microbial. 2: 542-554.

Vandenberghe, J., Thompson, F.L., Gomez-Gil, B. & Swings, J. 2003. Phenotypic diversity amongst Vibrio isolates from marine aquaculture systems. Aquaculture 219: 9-20.

Venter, J.C., Remington, K., Heidelberg, J.F., Halpern, A.L., Rusch, D., Eisen, J.A., Wu, D., Paulsen, I., Nelson, K.E., Nelson, W., Fouts, D.E., Levy, S., Knap, A.H., Lomas, M.W., Nealson, K., White, O., Peterson, J., Hoffman, J., Parsons, R., Baden-Tillson, H., Pfannkoch, C., Roger, Y.H. & Smith, H.O. 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66-74.

Veronica, A. 2005. Coastal Environmental Quality Initiative. http://repositoories.cdlib. /ucmarine/ceqi/009. Assessed on 9 July 2005.

Wang, C., Dang, H. & Ding, Y. 2008. Incidence of diverse integrons and β-lactamase genes in environmental Enterobacteriaceae isolates from Jiaozhou Bay, China. World J. Microbiol. Biotechnol. 24: 2889-2896.

Wright, A.C., Hill, R.T., Johnson, J.A., Roghman, M.C., Colwell, R.R. & Morris, J.G. Jr. 1996. Distribution of Vibrio vulnificus in the Chesapeake Bay. Applied and Environmental Microbiology 62: 717-724.

*Pengarang untuk surat-menyurat; email: drasmart@gmail.com