The Prevalence of Vibrio cholerae and Vibrio parahaemolyticus Virulence Genes and Multiple Antibiotics Resistant (MAR) Assessment from Local Shrimp Farm in Sarawak

Virulence and MAR index of Vibrio species

  • DAYANG NAJWA AWG BAKI Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
  • ELEXSON NILLIAN Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
  • DALENE LESEN Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Keywords: Antibiotic resistance, shrimp farm, Vibrio cholerae, V. parahaemolyticus, virulence genes


Excessive and improper antibiotic use in animals raised for human consumption can increase the risk of antibiotic-resistant infections, causing more harm and higher treatment costs. This study examined the virulence genes and antibiotic susceptibility of Vibrio cholerae and V. parahaemolyticus, two bacteria that can affect public health. A total of 32 water samples were collected from August to December 2021 from a shrimp farm in Sarawak. Ten (n = 10) V. cholerae and ten (n = 10) V. parahaemolyticus presumptive isolates were identified and purified using selective agar and duplex-PCR method. The results showed that 70% of V. cholerae isolates contained rtxA and 90% of V. cholerae isolates contained rtxC while tdh and trh were not found in V. parahaemolyticus isolates. Antibiotic susceptibility testing showed that all V. cholerae and V. parahaemolyticus isolates were resistant to at least one antibiotic with the mean Multiple Antibiotic Resistance (MAR) indices of 0.34 for V. cholerae and 0.24 for V. parahaemolyticus. The MAR index of 0.20 and greater indicates that antibiotics are heavily contaminating the shrimp farm water. This study highlights the need for the proper administration of antibiotics in shrimp farming environments to reduce the risk of antibiotic-resistant infections caused by V. cholerae and V. parahaemolyticus. Water treatment should also be implemented before being released back to the environment to lessen the negative impact brought by the rearing of shrimp from a highly contaminated source.


Author Biographies

ELEXSON NILLIAN, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia



DALENE LESEN, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia




Adesiyan, I.M., Bisi-Johnson, M.A., Ogunfowokan, A.O. & Okoh, A.I. (2021). Occurrence and antibiogram signatures of some Vibrio species recovered from selected rivers in South West Nigeria. Environmental Science and Pollution Research, 28(31): 42458-42476. 1007/s11356-021-13603-4

Adesiyan, I.M., Bisi-Johnson, M.A. & Okoh, A.I. (2022). Incidence of antibiotic resistance genotypes of Vibrio species recovered from selected freshwaters in Southwest Nigeria. Scientific Reports, 12(1): 18912. /s41598-022-23479-0

Broberg, C.A., Calder, T.J. & Orth, K. (2011). Vibrio parahaemolyticus cell biology and pathogenicity determinants. Microbes and Infection, 13(12-13): 992-1001. 06.013

Cabello, F.C. (2006). Heavy use of prophylactic antibiotics in aquaculture: A growing problem for human and animal health and for the environment. Environmental Microbiology, 8(7): 1137-1144.

Chen, L., Fan, J., Yan, T., Liu, Q., Yuan, S., Zhang, H., Yang, J., Deng, D., Huang, S. & Ma, Y. (2019). Isolation and characterization of specific phages to prepare a cocktail preventing Vibrio sp. Va-F3 infections in shrimp (Litopenaeus vannamei). Frontiers in Microbiology, 10: 2337. https://doi. org/10.3389/fmicb.2019.02337

Chow, K.H., Ng, T.K., Yuen, K.Y. & Yam, W.C. (2001). Detection of RTX toxin gene in Vibrio cholerae by PCR. Journal of Clinical Microbiology, 39(7): 2594–2597. 39.7.2594-2597.2001

CLSI. (2015). Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria. 3rd ed. CLSI guideline M45. Retrieved from treat/md_labs_ef39a/files/CLSI-M45ed3e-2018(1) .pdf

Coote, J.G. (1992). Structural and functional relationships among the RTX toxin determinants of Gram-negative bacteria. FEMS Microbiology Reviews, 8(2): 137-161. /j.1574-6968.1992.tb04961.x

Davis, R. & Brown, P.D.Y. (2016). Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica. Journal of Medical Microbiology, 65(4): 261-271.

Dell’Annunziata, F., Folliero, V., Giugliano, R., De Filippis, A., Santarcangelo, C., Izzo, V., Daglia, M., Galdiero, M., Arciola, C.R. & Franci, G. (2021). Gene transfer potential of outer membrane vesicles of Gram-negative bacteria. International Journal of Molecular Sciences, 22(11): 5985. /10.3390/ijms22115985

Deng, Y., Xu, H., Su, Y., Liu, S., Xu, L., Guo, Z., Wu, J., Cheng, C. & Feng, J. (2019). Horizontal gene transfer contributes to virulence and antibiotic resistance of Vibrio harveyi 345 based on complete genome sequence analysis. BMC Genomics, 20: 761.

Dick, M.H., Guillerm, M., Moussy, F. & Chaignat, C.L. (2012). Review of two decades of cholera diagnostics – How far have we really come? PLOS Neglected Tropical Diseases, 6(10): e1845.

Elexson, N., Afsah-Hejri, L., Rukayadi, Y., Soopna, P., Lee, H.Y., Tuan Zainazor, T.C., Nor Ainy, M., Nakaguchi, Y., Mitsuaki, N. & Son, R. (2014). Effect of detergents as antibacterial agents on biofilm of antibiotics-resistant Vibrio parahaemolyticus isolates. Food Control, 35(1): 378-385. .07.020

Farooq, A. & Unno, T. (2018). Plasmid profiling of multi-drug resistant Vibrio sp. isolated from influent and effluent water samples of fish farms in Jeju, South Korea. Korean Journal of Microbiology, 54(1): 53-59. 7095

Fathy Mahmoud, F. (2015). Effect of cooking methods on antibiotic residues in broiler chicken meat. Conference: 2nd International conference of Food Safety, Suez Canal University At: Faculty of Veterinary Medicin- Suez Canal University, 1(2015), 76-81. publication/322631993_Effect_of_Cooking_Meth ods_on_Antibiotic_Residues_in_Broiler_Chicken_Meat

Fisheries Development Authority of Malaysia (2020). Fisheries Development Authority of Malaysia. Annual Report.

Fletcher, S. (2015). Understanding the contribution of environmental factors in the spread of antimicrobial resistance. Environmental Health and Preventive Medicine, 20(4): 243-252. /s12199-015-0468-0

Fu, S., Hao, J., Jin, S., Wu, K., Wang, Y., Ye, S., Liu, Y. & Li, R. (2019). A human intestinal infection caused by a novel non-O1/O139 Vibrio cholerae genotype and its dissemination along the river. Frontiers in Public Health, 7(100): 1-6.

Fu, H., Yu, P., Liang, W., Kan, B., Peng, X. & Chen, L. (2020). Virulence, resistance, and genomic fingerprint traits of Vibrio cholerae isolated from 12 species of aquatic products in Shanghai, China. Microbial Drug Resistance, 26(12): 1526-1539.

Haifa-Haryani, W.O., Amatul-Samahah, M.A., Azzam-Sayuti, M., Chin, Y.K., Zamri-Saad, M., Natrah, I., Amal, M.N.A., Satyantini, W.H. & Ina-Salwany, M.Y. (2022). Prevalence, antibiotics resistance and plasmid profiling of Vibrio spp. isolated from cultured shrimp in Peninsular Malaysia. Microorganisms, 10(9): 1851. 3390/microorganisms10091851

Hassali, M.A.A., Yann, H.R., Verma, A.K., Hussain, R. & Sivaraman, S. (2018). Antibiotic use in food animals: Malaysia overview. School of Pharmaceutical Sciences Universiti Sains Malaysia, 33.

Holmström, K., Gräslund, S., Wahlström, A., Poungshompoo, S., Bengtsson, B.E. & Kautsky, N. (2003). Antibiotic use in shrimp farming and implications for environmental impacts and human health. International Journal of Food Science and Technology, 38: 255-266. /j.1365-2621.2003.00671.x

Hossain, M.M.M., Uddin, M.I., Islam, H., Fardoush, J., Rupom, M.A.H., Hossain, M.M., Farjana, N., Afroz, R., Hasan-Uj-Jaman, Roy, H.S., Shehab, M.A.S. & Rahman, M.A. (2020). Diagnosis, genetic variations, virulence, and toxicity of AHPND-positive Vibrio parahaemolyticus in Penaeus monodon. Aquaculture International, 28(6): 2531-2546.

Igere, B.E., Okoh, A.I. & Nwodo, U.U. (2022). Atypical and dual biotypes variant of virulent SA-NAG-Vibrio cholerae: An evidence of emerging/evolving patho-significant strain in municipal domestic water sources. Annals of Microbiology, 72(1): 1-13. s13213-021-01661-5

Joseph, T., Vaiyapuri, M., Reghunathan, D., Peeralil, Shaheer. P., Akhilnath, P.G. & Lalitha, K. (2015). Isolation and characterization of Vibrio cholerae O139 associated with mass mortality in Penaeus monodon and experimental challenge in postlarvae of three species of shrimp. Aquaculture, 442: 42-47.

Kriem, M.R., Banni, B., El Bouchtaoui, H., Hamama, A., El Marrakchi, A., Chaouqy, N., Robert-Pillot, A. & Quilici, M.L. (2015). Prevalence of Vibrio spp. in raw shrimps (Parapenaeus longirostris) and performance of a chromogenic medium for the isolation of Vibrio strains. Letters in Applied Microbiology, 61(3): 224-230. 1111/lam.12455

Kuthoose, M.F.A., Suhaimi, N.S.M., Ibrahim, D. & Zarkasi, K.Z. (2021). Microbial community diversity associated with healthy and unhealthy shrimp (early mortality syndrome) at Malaysian shrimp farm. Songklanakarin Journal of Science and Technology (SJST), 43: 897902. 10.14456/SJST-PSU.2021.118

Lee, B.C., Choi, S.H. & Kim, T.S. (2008). Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus. Microbes and Infection, 10(14): 1504-1513. .1016/j.micinf.2008.09.006

Lin, Q., Xavier, B.B., Alako, B.T.F., Mitchell, A.L., Rajakani, S.G., Glupczynski, Y., Finn, R.D., Cochrane, G. & Malhotra-Kumar, S. (2022). Screening of global microbiomes implies ecological boundaries impacting the distribution and dissemination of clinically relevant antimicrobial resistance genes. Communications Biology, 5(1): 1-9.

Linhartová, I., Bumba, L., Mašín, J., Basler, M., Osička, R., Kamanová, J., Procházková, K., Adkins, I., Hejnová-Holubová, J., Sadílková, L., Morová, J. & Šebo, P. (2010). RTX proteins: A highly diverse family secreted by a common mechanism. Fems Microbiology Reviews, 34(6): 1076-1112. https: //

Luu, Q.H., Nguyen, T.B.T., Nguyen, T.L.A., Do, T.T. T., Dao, T.H.T. & Padungtod, P. (2021). Antibiotics use in fish and shrimp farms in Vietnam. Aquaculture Reports, 20: 100711. https://doi. org/10.1016/j.aqrep.2021.100711

Madhusudana, R.B. & Surendran, P.K. (2013). Detection of ctx gene positive non-O1/non-O139 V. cholerae in shrimp aquaculture environments. Journal of Food Science and Technology, 50(3): 496-504.

Mandal, J., Sangeetha, V., Ganesan, V., Parveen, M., Preethi, V., Harish, B.N., Srinivasan, S. & Parija, S.C. (2012). Third-generation Cephalosporin–resistant Vibrio cholerae, India. Emerging Infectious Diseases, 18(8): 1326-1328. https://doi. org/10.3201/eid1808.111686

Manjusha, S. & Sarita, G.B. (2011). Plasmid associated antibiotic resistance in Vibrios isolated from coastal waters of Kerala. International Food Research Journal, 18(3): 1171-1181. http://www.ifrj.upm. df

Matsuda, S., Kodama, T., Okada, N., Okayama, K., Honda, T. & Iida, T. (2010). Association of Vibrio parahaemolyticus thermostable direct hemolysin with lipid rafts is essential for cytotoxicity but not hemolytic activity. Infection and Immunity, 78(2): 603-610.

Mrityunjoy, A., Kaniz, F., Fahmida, J., Shanzida, J.S., Md. Aftab, U. & Rashed, N. (2013). Prevalence of Vibrio cholerae in different food samples in the city of Dhaka, Bangladesh. International Food Research Journal. 20(2): 1017-1022. https://www.resea

Narayanan, S.V., Joseph, T.C., Peeralil, S., Koombankallil, R., Vaiyapuri, M., Mothadaka, M. P. & Lalitha, K.V. (2020). Tropical shrimp aquaculture farms harbour pathogenic Vibrio parahaemolyticus with high genetic diversity and Carbapenam resistance. Marine Pollution Bulletin, 160: 111551. .2020.111551

Ngo, H. & Fotedar, R. (2010). A review of probiotics in shrimp aquaculture. Journal of Applied Aquaculture, 22: 251-266. 10454438.2010.500597

Noorlis, A., Ghazali, F. & Cheah, Y.K. (2011). Antibiotic resistance and biosafety of Vibrio cholerae and Vibrio parahaemolyticus from freshwater fish at retail level. International Food Research Journal, 18(4): 1523-1530.

Osunla, C. & Okoh, A. (2017). Vibrio pathogens: A public health concern in rural water resources in sub-Saharan Africa. International Journal of Environmental Research and Public Health, 14(10): 1188.

Peng, X., Yu, K.Q., Deng, G.H., Jiang, Y.X., Wang, Y., Zhang, G.X. & Zhou, H.W. (2013). Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags. Journal of Microbiological Methods, 95(3): 455-462.

Pérez-Rodríguez, F. & Mercanoglu Taban, B. (2019). A state-of-art review on multidrug resistant pathogens in foods of animal origin: Risk factors and mitigation strategies. Frontiers in Microbiology, 10: 2091.

Prithvisagar, K.S., Krishna Kumar, B., Kodama, T., Rai, P., Iida, T., Karunasagar, I. & Karunasagar, I. (2021). Whole genome analysis unveils genetic diversity and potential virulence determinants in Vibrio parahaemolyticus associated with disease outbreak among cultured Litopenaeus vannamei (Pacific white shrimp) in India. Virulence, 12(1): 1936-1949. .2021.1947448

Raghunath, P. (2015). Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Frontiers in Microbiology, 5: 805. .2014.00805

Reda, A., Sah, R., Abdelaal, A., Shrestha, S. & Rodriguez-Morales, A.J. (2022). The emergence of cholera in multiple countries amidst current COVID-19 pandemic: Situation and implications for public health and travel medicine. Travel Medicine and Infectious Disease, 49: 102423.

Robert-Pillot, A., Guénolé, A., Lesne, J., Delesmont, R., Fournier, J.M. & Quilici, M.L. (2004). Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish colleted in two France coastal areas and from seafood imported into France. International Journal of Food Microbiology, 91: 319-325.

Said, B., Smith, H.R., Scotland, S.M. & Rowe, B. (1995). Detection and differentiation of the gene for toxin co-regulated pili (tcpA) in Vibrio cholerae non-O1 using the polymerase chain reaction. FEMS Microbiology Letters, 125: 5. 1016/0378-1097(94)00499-H

Sarjito, S. & Sabdono, A. (2021). Associated Vibrio species in shrimp Vibriosis from traditional brackish water pond in the north coastal of central Java, Indonesia. Genetics of Aquatic Organisms, 5(2): 45-54.

Satitsri, S., Pongkorpsakol, P., Srimanote, P., Chatsudthipong, V. & Muanprasat, C. (2016). Pathophysiological mechanisms of diarrhea caused by the Vibrio cholerae O1 El Tor variant: An in vivo study in mice. Virulence, 7(7): 789-805.

Shishehchian, F. (2020). Prevention and Solution for White Feces Disease (WFD) in Litopenaeus Vannamei Culture. Retrieved June 21, 2023 from _74709/prevention-and-solution-for-white-feces-di sease-wfd-in-litopenaeus-vannamei-culture-31e4d c393410

Takahashi, A., Kenjyo, N., Imura, K., Myonsun, Y. & Honda, T. (2000). Cl− secretion in colonic epithelial cells induced by the Vibrio parahaemolyticus hemolytic toxin related to thermostable direct hemolysin. Infection and Immunity, 68(9): 5435-5438. 10.1128/iai.68.9.5435-5438.2000

Ventola, C.L. (2015). The antibiotic resistance crisis. Pharmacy and Therapeutics, 40(4): 277-283.

Verma, J., Bag, S., Saha, B., Kumar, P., Ghosh, T.S., Dayal, M., Senapati, T., Mehra, S., Dey, P., Desigamani, A., Kumar, D., Rana, P., Kumar, B., Maiti, T.K., Sharma, N.C., Bhadra, R.K., Mutreja, A., Nair, G.B., Ramamurthy, T. & Das, B. (2019). Genomic plasticity associated with antimicrobial resistance in Vibrio cholerae. Proceedings of the National Academy of Sciences of the United States of America, 116(13): 6226-6231. DOI: 10.1073/pnas.1900141116

Vieira, R.H.S.F., Costa, R.A., Menezes, F.G.R., Silva, G.C., Theophilo, G.N.D., Rodrigues, D.P. & Maggioni, R. (2011). Kanagawa-negative, tdh- and trh-positive Vibrio parahaemolyticus isolated from fresh oysters marketed in Fortaleza, Brazil. Current Microbiology, 63(2): 126-130. 1007/s00284-011-9945-x

World Wildlife Fund. (2022). Farmed Shrimp Overview. World Wildlife Fund. Retrieved June 21, 2023 from industries/farmed-shrimp

Wu, Q., Vaziri, A.Z., Omidi, N., Hassan Kaviar, V., Maleki, A., Khadivar, P. & Kouhsari, E. (2023). Antimicrobial resistance among clinical Vibrio cholerae non-O1/non-O139 isolates: Systematic review and meta-analysis. Pathogens and Global Health, 117(3): 235-244. 20477724.2022.2114620

Xu, M., Wu, J. & Chen, L. (2019). Virulence, antimicrobial and heavy metal tolerance, and genetic diversity of Vibrio cholerae recovered from commonly consumed freshwater fish. Environmental Science and Pollution Research International, 26(26): 27338-27352. 10.1007/s11356-019-05287-8

Yano, Y., Hamano, K., Satomi, M., Tsutsui, I., Ban, M. & Aue-umneoy, D. (2014). Prevalence and antimicrobial susceptibility of Vibrio species related to food safety isolated from shrimp cultured at inland ponds in Thailand. Food Control, 38: 30-36.

Zhang, Y.B., Li, Y. & Sun, X.L. (2011). Antibiotic resistance of bacteria isolated from shrimp hatcheries and cultural ponds on Donghai Island, China. Marine Pollution Bulletin, 62(11): 2299-2307. 048

Zoqratt, M.Z.H.M., Eng, W.W.H., Thai, B.T., Austin, C.M. & Gan, H.M. (2018). Microbiome analysis of Pacific white shrimp gut and rearing water from Malaysia and Vietnam: Implications for aquaculture research and management. PeerJ, 6: e5826. https://

How to Cite
DAYANG NAJWA AWG BAKI, ELEXSON NILLIAN, & DALENE LESEN. (2023). The Prevalence of Vibrio cholerae and Vibrio parahaemolyticus Virulence Genes and Multiple Antibiotics Resistant (MAR) Assessment from Local Shrimp Farm in Sarawak. Borneo Journal of Resource Science and Technology, 13(1), 93-107.