Isolation, molecular identification, and pathogenicity of Aeromonas hydrophila disease in clown knife fish (Chitala chitala) in the Mekong Delta, Vietnam https://doi.org/10.12982/VIS.2025.010
Article Sidebar
Main Article Content
Abstract
Hemorrhagic septicemia disease caused by Aeromonas hydrophila bacteria appears to be more common and causes severe damage to many fish species worldwide. This research aimed to isolate, identify, and determine the pathogenicity and antibiotic susceptibility of A. hydrophila in septicemic clown knife (Chitala chitala) fish in the Mekong Delta, Vietnam. As a result, 116 bacterial strains were isolated from 176 septicemic fish samples. Two strains of bacteria, D2F71 and H1F39, were identified as A. hydrophila based on colony morphology, identification results by the API 20E kit, PCR technique, and aerolysin gene sequencing. In addition, these two strains of bacteria were experimentally infected on healthy clown knife fish (weight 15±4 g/fish) by injection method. The experimental results fulfilled Koch's postulates: infected fish showed signs of disease symptoms similar to those of naturally diseased fish. In particular, the two strains of bacteria D2F71 and H1F39 in the experiment had relatively high virulence, with LD50 values of 1.26×104 CFU/mL and 4.06×103 CFU/mL, respectively. The obtained findings demonstrated that the bacteria were susceptible to doxycycline, florfenicol, cefotaxime, flumequine, enrofloxacin, and sulfamethoxazole-trimethoprim at a rate of over 70%. On the contrary, the study found that bacterial isolates were resistant to ampicillin (100%), cefazolin (85%), streptomycin (80%), and colistin (80%). To our knowledge, this is the first report on hemorrhagic septicemic clown knife fish caused by A. hydrophila in Vietnam.
Article Details
This work is licensed under a Creative Commons Attribution 4.0 International License.
Publishing an article with open access in Veterinary Integrative Sciences leaves the copyright with the author. The article is published under the Creative Commons Attribution License 4.0 (CC-BY 4.0), which allows users to read, copy, distribute and make derivative works from the material, as long as the author of the original work is cited.
References
Abolghait, S.K., Akeda, Y., Kodama, T., Cantarelli, V.V., Iida, T., Honda, T., 2010. Aeromonas hydrophila PepO outer membrane endopeptidase activates human big endothelin-3 in vitro and induces skin ulcer in goldfish (Carassius auratus). Vet. Microbiol. 145(1-2), 113-121.
Ali, S., Akhter, S., Muhammad, A., Khan, I., Khan, W.A., Iqbal, M.N., Ali, Q., 2016. Identification, characterization and antibiotic sensitivity of Aeromonas hydrophila, a causative agent of epizootic ulcerative syndrome in wild and farmed fish from potohar, Pakistan. Pakistan. J. Zool. 48(3), 899-901.
Armwood, A.R., Griffin, M.J., Richardson, B.M., Wise, D.J., Ware, C., Camus, A.C., 2022. Pathology and virulence of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella anguillarum in channel (Ictalurus punctatus), blue (Ictalurus furcatus), and channel blue hybrid catfish. J. Fish. Dis. 45(11), 1683-1698.
Bannai, M.A.A., Kenani, A.J.K., Al Shammari, N.A., Al-Nagar, G., Mustafa, F., 2020. Isolation and treatments of Aeromonas hydrophila and Staphylococcus lentus implicated in the seasonal autumn mortalities of farm-raised Cyprinus carpio, Basrah governorate, Iraq. J. Phys. Conf. Ser. 1660, 012004.
Barrow, G.I., Feltham, R.K.A., 2004. Motility test. Cowan and steel’s manual for the identification of medical bacteria, 3rd edition. Cambridge University Press, UK.
Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493-496.
Buller, N.B., 2014. Bacteria and fungi from fish and other aquatic animals: a practical identification manual, 2nd edition. CABI, Wallingford.
Busse, H.J., Denner, E.B., Lubitz, W., 1996. Classification and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics. J. Biotechnol. 47(1), 3-38.
CLSI, 2020. Performance standards for antimicrobial susceptibility testing for bacteria isolated from aquatic animals. In: CLSI supplement VET04, (3rd edition). Clinical and Laboratory Standards Institute, Wayne, PA.
Crumlish, M., Thanh, P.C., Koesling, J., Tung, V.T., Gravningen, K., 2010. Experimental challenge studies in Vietnamese catfish, Pangasianodon hypophthalmus (Sauvage), exposed to Edwardsiella ictaluri and Aeromonas hydrophila. J. Fish Dis. 33(9), 717-722.
Chau, D.T.M., Mai, L.H., Dung, T.T., 2018. Antibiotic sensitivity of visceral white spot bacteria in snakehead (Channa striata) in Tra Vinh. CTUJoS. 54, 108-115.
Dahdouh, B., Basha, O., Khalil, S., Tanekhy, M., 2016. Molecular characterization, antimicrobial susceptibility and salt tolerance of Aeromonas hydrophila from fresh, brackish and marine fishes. Alex. J. Vet. Sci. 48(2), 46-53.
Dang, L.E., Nguyen, L.H.T., Vo, C.D., Bui, V.H.T., Nguyen, L.V., Phan, V.T., 2020. Status of Vietnam's national action plan on antimicrobial resistance in aquaculture. Asian. Fish. Sci. 33, 112-118.
Delghandi, M.R., El-Matbouli, M., Menanteau-Ledouble, S., 2020. Mycobacteriosis and infections with non-tuberculous mycobacteria in aquatic organisms: a review. Microorganisms. 8(9), 1368.
Dias, M.K., Sampaio, L.S., Proietti-Junior, A.A., Yoshioka, E.T., Rodrigues, D.P., Rodriguez, A.F., Tavares-Dias, M., 2016. Lethal dose and clinical signs of Aeromonas hydrophila in Arapaima gigas (Arapaimidae), the giant fish from Amazon. Vet. Microbiol. 188, 12-15.
Didugu, H., Krishnaiah, N., Rao, M.T., Ramanipushpa, R.N., 2016. Antibiogram of Aeromonas species isolated from livestock products. Int. J. Sci. Environ. Technol. 5(2), 475-478.
Dubey, S., Ager-Wick, E., Peng, B., Evensen, Ø., Sørum, H., Munangandu, H.M., 2022. Characterization of virulence and antimicrobial resistance genes of Aeromonas media strain SD/21-15 from marine sediments in comparison with other Aeromonas spp. Front. Microbiol. 13, 1022639.
Duc, P.M., Thy, T.N.T., Hatai, K., 2013. Pathogenicity of Aeromonas hydrophila to snakehead (Channa striata) fingerlings. Fish. Pathol. 48, 48-51.
Duman, M., Mulet, M., Altun, S., Saticioglu, I.B., Ozdemir, B., Ajmi, N., García-Valdés, E., 2021. The diversity of Pseudomonas species isolated from fish farms in Turkey. Aquac. 535, 736369.
Eid, H.M., El-Mahallawy, H.S., Shalaby, A.M., Elsheshtawy, H.M., Shetewy, M.M., Eidaroos, N.H., 2022. Emergence of extensively drug-resistant Aeromonas hydrophila complex isolated from wild Mugil cephalus (striped mullet) and Mediterranean seawater. Vet. World. 15(1), 55.
Frerichs, G.N., Millar, S.D., 1993. Mannual for the isolate and indentification of fish bacterial pathogens. Institute of Aquaculture, University of Stirling, Scotland.
Gam, L.T.H., Jensen, F.B., Damsgaard, C., Huong, D.T.T., Phuong, N.T., Bayley, M., 2001. Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity. Aquat. Toxicol. 187, 9-17.
Gulla, S., Bayliss, S., Björnsdóttir, B., Dalsgaard, I., Haenen, O., Jansson, E., Colquhoun, D.J., 2019. Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping. FEMS Microbiol. Lett. 366(7), fnz074.
Guo, P., Wang, N., Liu, Y., Lu, C., 2014. Antimicrobial susceptibility and characterization of outer membrane proteins of Aeromonas hydrophila isolated in China. J. Integr. Agric. 13(4), 911-917.
Guz, L., Kozinska, A., 2004. Antibiotic susceptibility of Aeromonas hydrophila and A. sobria isolated from farmed carp (Cyprinus carpio L.). Bull. Vet. Inst. Pulawy. 48, 391-395.
Hafez, A.E.E., Darwish, W.S., Elbayomi, R.M., Hussein, M.A., El Nahal, S.M., 2018. Prevalence, antibiogram, and molecular characterization of Aeromonas hydrophila isolated from frozen fish marketed in Egypt. Slov. Vet. Res. 55, 445-454.
Jacobs, L., Chenia, H.Y., 2007. Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems. Int. J. Food. Microbiol. 114(3), 295-306.
Jalal, K.C.A., Nur, Fatin, U.T., Mardiana, M.A., Akbar John, B., Kamaruzzaman, Y.B., Shahbudin, S., Muhammad, N.O., 2010. Antibiotic resistance microbes in tropical mangrove sediments in east coast peninsular, Malaysia. Afr. J. Microbiol. Res. 4(8), 640-645.
Janda, J.M., Abbott. S,L., 2010. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin. Microbiol. Rev. 23(1), 35-73.
Jin, L., Chen, Y., Yang, W., Qiao, Z., Zhang, X., 2020. Complete genome sequence of fish-pathogenic Aeromonas hydrophila HX-3 and comparative analysis: insights into virulence factors and quorum sensing. Sci. Rep. 10(1), 15479.
Kaskhedikar, M., Chhabra, D., 2010. Multiple drug resistance in Aeromonas hydrophila isolates of fish. Food. Microbiol. 28, 157-168.
Lee, B.H., Nicolas, P., Saticioglu, I.B., Fradet, B., Bernardet, J.F., Rigaudeau, D., Duchaud, E., 2023. Investigation of the genus Flavobacterium as a reservoir for fish-pathogenic bacterial species: the case of Flavobacterium collinsii. Appl. Environ. Microbiol. 89(4), e02162-22.
Lee, S.W., Wendy, W., 2017. Antibiotic and heavy metal resistance of Aeromonas hydrophila and Edwardsiella tarda isolated from red hybrid tilapia (Oreochromis spp.) coinfected with motile aeromonas septicemia and edwardsiellosis. Vet. World. 10(7), 803.
Manchanayake, T., Salleh, A., Amal, M.N.A., Yasin, I.S.M., Zamri-Saad. M., 2023. Pathology pathogenesis of Vibrio infection in fish: A review. Aquac. Rep. 28, 101459.
Masters, P., O’Bryan, T.A., Zurlo, J., Miller, D.Q., Joshi, N., 2003. Trimethoprim-sulfamethoxazole revisited. Arch. Intern. Med. 163, 402-410.
Miranda, C.D., Rojas, R., 2007. Occurrence of florfenicol resistance in bacteria associated with two Chilean salmon farms with different histories of antibacterial usage. Aquac. 266(1-4), 39-46.
Mulia, D.S., Isnansetyo, A., Pratiwi, R., Asmara, W., 2021. Antibiotic resistance of Aeromonas spp. isolated from diseased walking catfish (Clarias sp.). Biodiversitas. 22, 4839-4846.
Newman, S.G., 1993. Bacterial vaccines for fish. Annu. Rev. Fish Dis. 3, 145-185.
Noga, E.J., 2010. Fish disease: diagnosis and treatment. John Wiley & Sons, Hoboken, New Jersey.
Nguyen, H.N.K., Van, T.T.H., Nguyen, H.T., Smooker, P.M., Shimeta, J., Coloe, P.J., 2014. Molecular characterization of antibiotic resistance in Pseudomonas and Aeromonas isolates from catfish of the Mekong Delta, Vietnam. Vet. Microbiol. 171(3-4), 397-405.
Nhinh, D.T., Le, D.V., Van, K.V., Huong, N.T.G., Dang, L.T., Hoai, T.D., 2021. Prevalence, virulence gene distribution, and alarming the multidrug resistance of Aeromonas hydrophila associated with disease outbreaks in freshwater aquaculture. Antibiotics. 10(5), 532.
Odeyemi, O.A., Ahmad, A., 2017. Antibiotic resistance profiling and phenotyping of Aeromonas species isolated from aquatic sources. Saudi. J. Biol. Sci. 24(1), 65-70.
Oliveira, S.T., Veneroni-Gouveia, G., Costa, M.M., 2012. Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish. Pesqui. Vet. Brasil. 32, 701-706.
Pauzi, N.A., Mohamad, N., Azzam-Sayuti, M., Yasin, I.S.M., Saad, M.Z., Nasruddin, N.S., Azmai, M.N.A., 2020. Antibiotic susceptibility and pathogenicity of Aeromonas hydrophila isolated from red hybrid tilapia (Oreochromis niloticus×Oreochromis mossambicus) in Malaysia. Vet. World. 13, 2166-2171.
Pollard, D.R., Johnson, W.M., Lior, H., Tyler, S.D., Rozee, K.R., 1990. Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction. J. Clin. Microbiol. 28(11), 2477-2481.
Preena, P.G., Dharmaratnam, A., Jayadradhan, R.K., Swaminathan, V.T.R., 2021. Plasmid‐mediated antimicrobial resistance in motile aeromonads from diseased Nile tilapia (Oreochromis niloticus). Aquac. Res. 52(1), 237-248.
Pridgeon, J.W., Klesius, P.H., 2011. Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009. Dis. Aquat. Org. 94(3), 249-253.
Pungpian, C., Lee, S., Trongjit, S., Sinwat, N., Angkititrakul, S., Prathan, R., Chuanchuen, R., 2021. Colistin resistance and plasmid-mediated mcr genes in Escherichia coli and Salmonella isolated from pigs, pig carcass and pork in Thailand, Lao PDR and Cambodia border provinces. J. Vet. Sci. 22(5), e68.
Pham, T.H.H., Kim, D.H., Quach, V.C.T., Nguyen, P.T., Nguyen, T.L., 2023. Prevalence and antibiotic resistance of Aeromonas schubertii causing internal white spot disease on snakehead fish, Channa striata, in the Mekong Delta, Vietnam. J. World. Aquac. Soc. 54(5), 1-17.
Phu, T.M., Phuong, N.T., Dung, T.T., Hai, D.M., Son, V.N., Rico, A., Dalsgaard, A., 2016. An evaluation of fish health‐management practices and occupational health hazards associated with Pangasius catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta, Vietnam. Aquac. Res. 47(9), 2778-2794.
Qu, F.T., Wang, W.Q., Qian, L.I.U., Zhou, H.J., Hu, J.R., Du, X.L., Meng, S., 2022. Genetic diversity, antibiotic resistance, and pathogenicity of Aeromonas species from food products in Shanghai, China. Biomed. Environ. Sci. 35(9), 842-853.
Ramírez-Bayard, I.E., Mejía, F., Medina-Sánchez, J.R., Cornejo-Reyes, H., Castillo, M., Querol-Audi, J., Martínez-Torres, A.O., 2023. Prevalence of Plasmid-associated tetracycline resistance genes in multidrug-resistant escherichia coli strains isolated from environmental, animal and human samples in Panama. Antibiotics. 12(2), 280.
Reed, L.J., Muench, H.A., 1938. Simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27(3), 493-497.
Revina, O., Avsejenko, J., Cirule, D., Valdovska, A., 2017. Antimicrobial resistance of Aeromonas spp. Isolated from the sea trout (Salmo trutta L.) in Latvia. Res. Rural. Dev. 1, 271-275.
Saavedra, M.J., Guedes-Novais, S., Alves, A., Rema, P., Tacão, M., Correia, A., Martínez-Murcia, A., 2004. Resistance to β-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss). Int. Microbiol. 7(3), 207-211.
Samal, S.K., Das, B.K., Pal, B., 2014. Isolation, biochemical characterization, antibiotic susceptibility study of Aeromonas hydrophila isolated from freshwater fish. Int. J. Curr. Microbiol. Appl. Sci. 3(12), 259-267.
Samayanpaulraj, V., Sivaramapillai, M., Palani, S.N., Govindaraj, K., Velu, V., Ramesh, U., 2020. Identification and characterization of virulent Aeromonas hydrophila Ah17 from infected Channa striata in river Cauvery and in vitro evaluation of shrimp chitosan. Food Sci. Nutr. 8(2), 1272-1283.
Sambrook, J., Fritsch, E.R., Maniatis, T., 1989. Molecular cloning: a laboratory manual, (2nd edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sebastião, F.A., Majolo, C., Martins, V.F.S., Boijink, C.D.L., Brandão, F.R, Pereira, S.L.A., Chagas, E.C., 2023. Antimicrobial resistance profile of Aeromonas spp. isolated from asymptomatic Colossoma macropomum cultured in the Amazonas State, Brazil. Brazil. Braz. J. Biol. 82, e260773.
Semwal, A., Kumar, A., Kumar, N.A., 2023. Review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture. Heliyon. 9, e14088.
Sherif, A.H., Kassab, A.S., 2023. Multidrug-resistant Aeromonas bacteria prevalence in Nile tilapia broodstock. BMC Microbiol. 23(1), 80.
Smith, P., 2008. Aquaculture and florfenicol resistance in Salmonella enterica Typhimurium DT104. Emerg. Infect. Dis. 14(8), 1327.
So, N.V., Tuan, L.A., 2022. Assessment of the cultural situation and farming area management of Natopterus chitala in Hau Giang Province on the basis of GIS application. Vietnam. J. Agric. Rural. Dev. 2(4), 81-88.
Soliman, M.K., 2022. Treatment of MAS caused by Aeromonas sobria in European seabass. Egypt. J. Aquat. Biol. Fish. 26(5), 535-556.
Soto-Rodriguez, S.A., Lozano-Olvera, R., Garcia-Gasca, M.T., Abad-Rosales, S.M., Gomez-Gil, B., Ayala-Arellano, J., 2018. Virulence of the fish pathogen Aeromonas dhakensis: genes involved, characterization and histopathology of experimentally infected hybrid tilapia. Dis. Aquat. Org. 129(2), 107-116.
Syrova, E., Kohoutova, L., Dolejska, M., Papezikova, I., Kutilova, I., Cizek, A., Palikova, M., 2018. Antibiotic resistance and virulence factors in mesophilic Aeromonas spp. from Czech carp fisheries. J. Appl. Microbiol. 125(6), 1702-1713.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30(12), 2725-9.
Tanaka, K.H., Vincent, A.T., Emond-Rheault, J.G., Adamczuk, M., Frenette, M., Charette, S.J., 2017. Plasmid composition in Aeromonas salmonicida subsp. salmonicida 01-B526 unravels unsuspected type three secretion system loss patterns. BMC. Genomics. 18(1), 1-12.
Tien, N.V., Vu, H.S., Luu, D.L., Le, V.K., 2012. Effects of feeds on growth performance and feed efficiency of clown featherback fish Chilata ornata (Gray, 1831) fingerling stage. J. Sci. Dev. 10(4), 640-647.
Thaotumpitak, V., Sripradite, J., Atwill, E.R., Jeamsripong, S., 2023. Emergence of colistin resistance and characterization of antimicrobial resistance and virulence factors of Aeromonas hydrophila, Salmonella spp. and Vibrio cholerae isolated from hybrid red tilapia cage culture. Peer J. 11, e14896.
Thi, Q.V.C., Nguyen, B.T., Tu, T.D., 2023. Antimicrobial resistance and the prevalence of integron in Aeromonas hydrophila from hemorrhagic diseased Pangasius catfish of the Mekong Delta. Vet. Integr. Sci. 21(2), 333-347.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic. Acids. Res. 25(24), 4876-4882.
Treves-Brown, K., 2000. Applied fish pharmacology: Aquaculture series volume 3, Kluwer Academic Publication, Netherlands.
Van, D.H., Soltani, M., Leitão, A., Shafiei, S., Asadi, S., Lymbery, A.J., Ringø, E., 2022. Streptococcosis a Re-emerging disease in aquaculture: significance and phytotherapy. Animals. 12(18), 2443.
Viet, T.V., 2015. Applications of GIS for evaluation of the current culture status of Clown knife fish (Chitala ornata) in Phung Hiep District, Hau Giang Province. CTUJoS. 38, 109-115.
Xue, M., Xiao, Z., Li, Y., Jiang, N., Liu, W., Meng, Y., Zhou, Y., 2022. Isolation, identification and characteristics of Aeromonas caviae from diseased largemouth bass (Micropterus salmoides). Fishes. 7(3), 119.
Yang, Y., Miao, P., Li, H., Tan, S., Yu, H., Yu, H., 2018. Antibiotic susceptibility and molecular characterization of Aeromonas hydrophila from grass carp. J. Food Saf. 38(1), e12393.
Zdanowicz, M., Mudryk, Z.J., Perliński, P., 2020. Abundance and antibiotic resistance of Aeromonas isolated from the water of three carp ponds. Vet. Res. Commun. 44(1), 9-18.
Zhang, X.J., Qin, G.M., Bing, X.W., Yan, B.L., Bi, K.R., 2011. Phenotypic and molecular characterization of Photobacterium damselae, a pathogen of the cultured tongue sole Cynoglossus semilaevis in China. N. Z. J. Mar. Freshwater. Res. 45(1), 1-13.
Zheng, W., Cao, H., Yang, X., 2012. Grass carp (Ctenopharyngodon idellus) infected with multiple strains of Aeromonas hydrophila. Afr. J. Microbiol. Res. 6(21), 4512-4520.
Zhu, X., Qian, Q., Wu, C., Zhu, Y., Gao, X., Jiang, Q., Zhang, X., 2022. Pathogenicity of Aeromonas veronii Causing Mass Mortality of Largemouth Bass (Micropterus salmoides) and Its Induced Host Immune Response. Microorganisms. 10(11), 2198.