Abstract
Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90–98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah+act+ alt+ascV+eno+lip+ast+ on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD50 for 9C strain was 6.81 × 104 CFU/mL and 7.62 × 105 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aguilera-Arreola MG, Hernandez-Rodriguez C, Zuniga G, Figueras MJ, Castro-Escarpulli G (2005) Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes. FEMS Microbiol Lett 242(2):231–240. https://doi.org/10.1016/j.femsle.2004.11.011
Albert MJ, Ansaruzzaman M, Talukder KA, Chopra AK, Kuhn I, Rahman M, Faruque ASG, Islam MS, Sack RB, Mollby R (2000) Prevalence of enterotoxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, and the environment. J Clin Microbiol 38(10):3785–3790. https://doi.org/10.1128/JCM.38.10.3785-3790.2000
Balsalobre LC, Dropa M, Matte GR, Matte MH (2009) Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei. J Water Health 7(4):685–691. https://doi.org/10.2166/wh.2009.082
Bhowmik P, Bag PK, Hajra TK, De R, Sarkar P, Ramamurthy T (2009) Pathogenic potential of Aeromonas hydrophila isolated from surface waters in Kolkata, India. J Med Microbiol 58(12):1549–1558. https://doi.org/10.1099/jmm.0.014316-0
Carnahan AM, Behram S, Joseph SW (1991) Aerokey Il: A flexible key for identifying clinical Aeromonas species. J Clin Microbiol 29(12):2843–2849. https://doi.org/10.1128/JCM.29.12.2843-2849.1991
Chopra AK, Houston CW (1999) Enterotoxins in Aeromonas associated gastroenteritis. Microb Infect 1(13):1129–1137. https://doi.org/10.1016/s1286-4579(99)00202-6
De Figueiredo J, Plumb JA (1977) Virulence of different isolates of Aeromonas hydrophila in channel catfish. Aquaculture 11(4):349–354. https://doi.org/10.1016/0044-8486(77)90084-9
Dias MK, Sampaio LS, Proietti-Junior AA, Yoshioka ET, Rodrigues DP, Rodriguez AF, Ribeiro RA, Faria FS, Ozorio RO, 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. https://doi.org/10.1016/j.vetmic.2016.04.001
Gavin R, Merino S, Altarriba M, Canals R, Shaw JG, Tomas JM (2003) Lateral flagella are required for increased cell adherence, invasion and biofillm formation by Aeromonas spp. FEMS Microbiol Lett 224(1):77–83. https://doi.org/10.1016/S0378-1097(03)00418-X
Grim CJ, Kozlova EV, Sha J, Fitts EC, Lier CJV, Kirtley ML, Joseph SJ, Read TD, Burd EM, Tall BD, Joseph SW, Horneman AJ, Chopra AK, Shak JR (2013) Characterization of Aeromonas hydrophila wound pathotypes by comparative genomic and functional analyses of virulence genes. mBio 4(2):00064–00013. https://doi.org/10.1128/mBio.00064-13
Karunasagar I, Rosalind GM, Rao GK (1989) Aeromonas hydrophila septicaemia of Indian major carps in some commercial fish farms of WestGodavari District, Andhra Pradesh. Curr Sci 58(18):1044–1045
Khajanchi BK, Sha J, Kozlova EV, Erova TE, Suarez G, Sierra JC, Popov VL, Horneman AJ, Chopra AK (2009) N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiol 155(11):3518–3531. https://doi.org/10.1099/mic.0.031575-0
Kingombe CI, Huys G, Tonolla M, Albert MJ, Swings J, Peduzzi R, Jemmi T (1999) PCR detection, characterization, and distribution of virulence genes in Aeromonas spp. Appl Environ Microbiol 65(12):5293–5302. https://doi.org/10.1128/AEM.65.12.5293-5302.1999
Kirov SM (2003) Bacteria that express lateral flagella enable dissection of the multifunctional roles of flagella in pathogenesis. FEMS Microbiol Lett 224(2):151–159. https://doi.org/10.1016/S0378-1097(03)00445-2
Leung KY, Yeap IV, Lam TJ, Sin YM (1995) Serum resistance as a good indicator for virulence in Aeromonas hydrophila strains isolated from diseased fish in South- East Asia. J Fish Dis 18(6):511–518. https://doi.org/10.1111/j.1365-2761.1995.tb00355.x
Li J, Ni XD, Liu YJ, Lu CP (2011) Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish. J Appl Microbiol 110(3):823–830. https://doi.org/10.1111/j.1365-2672.2011.04944.x
Martino ME, Fasolato L, Montemurro F, Rosteghin M, Manfrin A, Patarnello T, Novelli E, Cardazzo B (2011) Determination of microbial diversity of Aeromonas strains on the basis of multilocus sequence typing, phenotype, and presence of putative virulence genes. Appl Environ Microbiol 77(14):4986–5000. https://doi.org/10.1128/AEM.00708-11
Mittal KR, Lalonde G, Leblanc D, Olivier G, Lallier R (1980) Aeromonas hydrophila in rainbow trout: relation between virulence and surface characteristics. Can J Microbiol 26(12):1501–1503. https://doi.org/10.1139/m80-248
Nawaz M, Khan SA, Khan AA, Sung K, Tran Q, Kerdahi K, Steele R (2010) Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiol 27(3):327–331. https://doi.org/10.1016/j.fm.2009.11.007
Palumbo SA, Maxino F, Williams AC, Buchanan RL, Thayer DW (1985) Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl Environ Microbiol 50(4):1027–1030. https://doi.org/10.1128/AEM.50.4.1027-1030.1985
Pillai L, Sha J, Erova TE, Fadl AA, Khajanchi BK, Chopra AK (2006) Molecular and functional characterization of a ToxR-regulated lipoprotein from a clinical isolate of Aeromonas hydrophila. Infect Immun 74(7):3742–3755. https://doi.org/10.1128/IAI.00402-06
Pridgeon JW, Klesius PH (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. https://doi.org/10.3354/dao02332
Rathore G, Swaminathan TR, Abidi R, Mahanta PC, Kapoor D (2005) Isolation and characterization of motile aeromonads from aquatic environment. Indian J Fish 52(2):241–248. https://doi.org/10.1017/S0950268800054121
Reed LJ, Munch H (1938) A simple method of estimating 50 percent and points. Am J Hyg 27:494. https://doi.org/10.1093/oxfordjournals.aje.a118408
Sachan N, Agarwal RK (2000) Selective enrichment broth for the isolation of Aeromonas sp. from chicken meat. Int J Food Microbiol 60(1):65–74. https://doi.org/10.1016/s0168-1605(00)00322-6
Sahoo PK, Swaminathan TR, Abraham TJ, Kumar R, Pattanayak S, Mohapatra A, Rath SS (2016) Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India. Acta Trop 161:8–17. https://doi.org/10.1016/j.actatropica.2016.05.004
Samayanpaulraj V, Velu V, Uthandakalaipandiyan R (2019) Determination of lethal dose of Aeromonas hydrophila Ah17 strain in snake head fish Channa striata. Microb Pathog 127:7–11. https://doi.org/10.1016/j.micpath.2018.11.035
Sen K, Rodgers M (2004) Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification. J Appl Microbiol 97(5):1077–1086. https://doi.org/10.1111/j.1365-2672.2004.02398.x
Seshadri R, Joseph SW, Chopra AK, Sha J, Shaw J, Graf J, Haft D, Wu M, Ren Q, Rosovitz MJ, Madupu R (2006) Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades. J Bacteriol 188(23):8272–8282. https://doi.org/10.1128/JB.00621-06
Sha J, Erova TE, Alyea RA, Wang S, Olano JP, Pancholi V, Chopra AK (2009) Surface- expressed enolase contributes to the pathogenesis of clinical isolate SSU of Aeromonas hydrophila. J Bacteriol 191(9):3095–3107. https://doi.org/10.1111/j.1567-1364.2012.00806.x
Sierra JC, Suarez G, Sha J, Foltz SM, Popov VL, Galindo CL, Garner HR, Chopra AK (2007) Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila—part II. Microbiol Pathog 43(4):147–160. https://doi.org/10.1016/j.micpath.2007.05.003
Thayumanavan T, Vivekanandhan G, Savithamani K, Subashkumar R, Lakshmanaperumalsamy P (2003) Incidence of haemolysin-positive and drug-resistant Aeromonas hydrophila in freshly caught finfish and prawn collected from major commercial fishes of coastal South India. FEMS Immunol Med Microbiol 36(1–2):41–45. https://doi.org/10.1016/S0928-8244(03)00037-3
Verma DK, Rathore G (2015) New host record of five Flavobacterium species associated with tropical fresh water farmed fishes from North India. Braz J Microbiol 46(4):969–976. https://doi.org/10.1590/S1517-838246420131081
Vilches S, Urgell C, Merino S, Chacon MR, Soler L, Castro-Escarpulli G, Figueras MJ, Tomas JM (2004) Complete type III secretion system of a mesophilic Aeromonas hydrophila strain. Appl Environ Microbiol 70(11):6914–6919. https://doi.org/10.1128/AEM.70.11.6914-6919.2004
Vilches S, Wilhelms M, Yu HB, Leung KY, Tomas JM, Merino S (2008) Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system. Microb Pathog 44(1):1–12. https://doi.org/10.1016/j.micpath.2007.06.004
Vivekanandhan G, Hatha AAM, Lakshmanaperumalsamy P (2005) Prevalence of Aeromonas hydrophila in fish and prawns from the sea food market of Coimbatore, South India. Food Microbiol 22:133–137. https://doi.org/10.1016/j.fm.2004.01.015
Wang G, Clark CG, Liu C, Pucknell C, Munro CK, Kruk TMAC, Caldeira R, Woodward DL, Rodgers FG (2003) Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by Multiplex PCR. J Clinical Microbiol 41(3):1048–1054. https://doi.org/10.1128/JCM.41.3.1048-1054.2003
Yadav S, Verma DK, Pradhan PK, Dobriyal AK, Sood N (2014) Phenotypic and genotypic identification of Aeromonas species from aquatic environment. Int J Aquat Sci 5(1):3–20
Yu HB, Rao PS, Lee HC, Vilches S, Merino S, Tomas JM, Leung KY (2004) A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect Immun 72(3):1248–1256. https://doi.org/10.1128/iai.72.3.1248-1256.2004
Zhou Y, Fan Y, Jiang N, Liu W, Shi Y, Zhao J, Zeng L (2018) Molecular characteristics and virulence analysis of eight Aeromonas hydrophila isolates obtained from diseased Amur sturgeon Acipenser schrenckii Brandt, 1869. J Vet Med Sci 80(3):421–426. https://doi.org/10.1292/jvms.17-0529
Acknowledgements
The authors wish to express their sincere thanks to Director, ICAR-NBFGR, Lucknow, India for providing the facilities and financial help under the institute funded project FISHNBFGRIL201600. The first author is also thankful to Director, ICAR-CIFE, Mumbai, for their support as this work also forms part of her Ph. D thesis.
Funding
The present work was carried out under ICAR-NBFGR institute funded project FISHNBFGRIL201600.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest to publish this manuscript.
Rights and permissions
About this article
Cite this article
Muduli, C., Tripathi, G., Paniprasad, K. et al. Virulence potential of Aeromonas hydrophila isolated from apparently healthy freshwater food fish. Biologia 76, 1005–1015 (2021). https://doi.org/10.2478/s11756-020-00639-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.2478/s11756-020-00639-z