Bacterial diseases are common in ornamental fish, more frequently associated with ubiquitous bacteria from the aquarium environment. The disease can lead to fish mortality and cause high economic losses if not rapidly controlled. The aim of this study was to identify the main causative bacterial agents of infection in ornamental fish with different clinical signs. A total of 126 freshwater fish, from 12 families and 38 species, with clinical signs were collected in a wholesaler in São Paulo, SP, Brazil. Samples were taken from the eye, skin ulcers, kidneys, and gills, plated on MacConkey, CHROMagar Orientation, and blood agar and incubated under aerobic and anaerobic conditions. Bacterial identification was performed by MALDI-TOF mass spectrometry. From the 126 studied animals, 112 were positive for bacterial isolation. Among the positive animals, 32.1% presented infection caused by a single bacterial species, while in the remaining 67.9%, two to six different bacterial species were identified. A total of 259 bacterial strains were obtained and classified among 46 bacterial species. The species of higher frequency were Aeromonas veronii (26.3%), Aeromonas hydrophilla (16.2%), Shewanella putrefaciens (7.3%), Citrobacter freundii (8.1%), Vibrio albensis (5.8%), and Klebsiella pneumoniae (4.2%). MALDI-TOF MS showed to be a rapid method for diagnosis of bacterial disease outbreaks in ornamental fish establishments.

Centro Universitário Max Planck Indaiatuba Brazil

Companhia Ambiental do Estado de São Paulo São Paulo Brazil

Departamento de Medicina Veterinária Preventiva e Saúde Animal Faculdade de Medicina Veterinária Universidade de São Paulo Professor Dr Orlando Marques Paiva São Paulo SP CEP 05508270 Brazil

See more in PubMed

ABINPET (2019) Dados de Mercado [Internet]. Brazil: Associação Brasileira da Indústria de produtos para Animais de Estimação; http://abinpet.org.br/mercado/ . Accessed 25 Oct 2020

Abraham TJ, Paul P, Adikesavalu H, Patra A, Banerjee S (2016) Stenotrophomonas maltophilia as an opportunistic pathogen in cultured African catfish Clarias gariepinus (Burchell, 1822). Aquaculture 450:168–172. https://doi.org/10.1016/j.aquaculture.2015.07.015 DOI

Acha PN, Szyfres B (2003) Zoonoses and communicable diseases common to man and animals. Bacterioses and Mycoses, vol. I., 3rd Ed. Pan American Health Organization, Regional Office of the WHO, Washington

Al-Harbi AH, Uddin MN (2004) Seasonal variation in the intestinal bacterial flora of hybrid tilapia (Oreochromis niloticus x Oreochromis aureus) cultured in earthen ponds in Saudi Arabia. Aquaculture 229:37–44. https://doi.org/10.1016/S0044-8486(03)00388-0 DOI

Altun S, Gül Büyükekiz A, Duman M et al (2014) Isolation of Shewanella putrefaciens from goldfish (Carassius auratus). Isr J Aquacult Bamidgeh 66

Bandeira Junior G, dos Santos AC, Souza CF et al (2018) Citrobacter freundii infection in silver catfish (Rhamdia quelen): hematological and histological alterations. Microb Pathog 125:276–280. https://doi.org/10.1016/j.micpath.2018.09.038 PubMed DOI

Beaz-Hidalgo R, Agüeria D, Latif-Eugenín F, Yeannes MI, Figueras MJ (2015) Molecular characterization of Shewanella and Aeromonas isolates associated with spoilage of common carp (Cyprinus carpio). FEMS Microbiol Lett 362(1):1–8. https://doi.org/10.1093/femsle/fnu029 PubMed DOI

Behera BK, Bera AK, Paria P, Das A, Parida PK, Kumari S, Bhowmick S, Das BK (2018) Identification and pathogenicity of Plesiomonas shigelloides in silver carp. Aquaculture 493:314–318. https://doi.org/10.1016/j.aquaculture.2018.04.063 DOI

Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-Van Dillen PME, Van Der Noordaa J (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28:495–503 DOI

Bretzinger A, Fisher-Scherl T, Oumouna M et al (1999) Mass mortalities in koi carp, Cyprinus carpio, associated with gill and skin disease. Bull Eur Assoc Fish Pathol 19:182–199

Burbick CR, Nydam SD, Hendrix GK, Besser TE, Diaz D, Snekvik K (2018) Use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of pathogenic Vibrio in fish. J Aquat Anim Health 30:332–338. https://doi.org/10.1002/aah.10044 PubMed DOI

Chen YS, Liu YC, Yen MY, Wang JH, Wang JH, Wann SR, Cheng DL (1997) Skin and soft-tissue manifestations of Shewanella putrefaciens infection. Clin Infect Dis 25(2):225–229. https://doi.org/10.1086/514537 PubMed DOI

Citarasu T, Alfred Dhas K, Velmurugan S et al (2011) Isolation of Aeromonas hydrophila from infected ornamental fish hatchery during massive outbreaks. Int J Curr Res 2(1):37–41

Davis KB, Griffin BR, Gray WL (2002) Effect of handling stress on susceptibility of channel catfish Ictalurus punctatus to Ichthyophthirius multifiliis and channel catfish virus infection. Aquaculture 214(1–4):55–66. https://doi.org/10.1016/S0044-8486(02)00362-9 DOI

Dobiasova H, Kutilova I, Piackova V, Vesely T, Cizek A, Dolejska M (2014) Ornamental fish as a source of plasmid-mediated quinolone resistance genes and antibiotic resistance plasmids. Vet Microbiol 171(3–4):413–421. https://doi.org/10.1016/j.vetmic.2014.02.011 PubMed DOI

El-Jeni R, El Bour M, Calo-Mata P et al (2016) In vitro probiotic profiling of novel Enterococcus faecium and Leuconostoc mesenteroides from Tunisian freshwater fish. Can J Microbiol 62(1):60–71. https://doi.org/10.1139/cjm-2015-0481 PubMed DOI

Gallani SU, Sebastião FA, Valladão GMR et al (2016) Pathogenesis of mixed infection by Spironucleus sp. and Citrobacter freundii in freshwater angelfish Pterophyllum scalare. Microb Pathog 100:119–123. https://doi.org/10.1016/j.micpath.2016.09.002 PubMed DOI

Gauthier DT (2015) Bacterial zoonoses of fish: a review and appraisal of evidence for linkages between fish and human infections. Vet J 203(1):27–35. https://doi.org/10.1016/j.tvjl.2014.10.028 PubMed DOI

Geng Y, Wang K, Chen D, Huang X, He M, Yin Z (2010) Stenotrophomonas maltophilia, an emerging opportunist pathogen for cultured channel catfish, Ictalurus punctatus, in China. Aquaculture 308(3–4):132–135. https://doi.org/10.1016/j.aquaculture.2010.08.032 DOI

Grim CJ, Kozlov EV, Sha J, Fitts EC et al (2013) Characterization of Aeromonas hydrophila wound pathotypes by comparative genomic and functional analyses of virulence genes. mBio 4(2):1–13. https://doi.org/10.1128/mBio.00064-13 DOI

Grutter AS, Pankhurst NW (2000) The effects of capture, handling, confinement and ectoparasite load on plasma levels of cortisol, glucose and lactate in the coral reef fish Hemigymnus melapterus. J Fish Biol 57(2):391–401. https://doi.org/10.1006/jfbi.2000.1312 DOI

Hijazin M, Alber J, Lämmler C, Weitzel T, Hassan AA, Timke M, Kostrzewa M, Prenger-Berninghoff E, Zschöck M (2012) Identification of Trueperella (Arcanobacterium) bernardiae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis and by species-specific PCR. J Med Microbiol 61(3):457–459. https://doi.org/10.1099/jmm.0.035774-0 PubMed DOI

Holt HM, Gahrn-Hansen B, Bruun B (2005) Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics. Clin Microbiol Infect 11(5):347–352. https://doi.org/10.1111/j.1469-0691.2005.01108.x PubMed DOI

Honein K, Jagoda SSSS, Arulkanthan A et al (2018) Draft genome sequence of Aeromonas hydrophila strain Ae25, isolated from a septicemic moribund koi carp (Cyprinus carpio) in Sri Lanka. Genome Announc 6(5):e01523–e01517. https://doi.org/10.1128/genomeA.01523-17 PubMed DOI PMC

Janda JM (2014) Shewanella: a marine pathogen as an emerging cause of human disease. Clin Microbiol Newsl 36(4):25–29. https://doi.org/10.1016/j.clinmicnews.2014.01.006 DOI

Kačániová M, Klūga A, Kántor A, Medo J, Žiarovská J, Puchalski C, Terentjeva M (2019) Comparison of MALDI-TOF MS Biotyper and 16S rDNA sequencing for the identification of Pseudomonas species isolated from fish. Microb Pathog 132:313–318. https://doi.org/10.1016/j.micpath.2019.04.024 PubMed DOI

Kazazić SP, Topić Popović N, Strunjak-Perović I, Babić S, Florio D, Fioravanti M, Bojanić K, Čož-Rakovac R (2019) Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream. PLoS One 14:e0225343. https://doi.org/10.1371/journal.pone.0225343 PubMed DOI PMC

Khashe S, Janda JM (1998) Biochemical and pathogenic properties of Shewanella alga and Shewanella putrefaciens. J Clin Microbiol 36(3):783–787 DOI

Koziñska A, Pêkala A (2004) First isolation of Shewanella putrefaciens from freshwater fish - a potential new pathogen of fish. Bull Eur Ass Fish Patholl 24(4):199–203

Ksenija A, Jelena A, Dušan M et al (2016) Differentiation between Pseudomonas and Stenotrophomonas species isolated from fish using molecular and MALDI-TOF method. Acta Vet 66(3):304–316. https://doi.org/10.1515/acve-2016-0027 DOI

Kusdarwati R, Rozi, Dinda D et al (2018) Antimicrobial resistance prevalence of Aeromonas hydrophila isolates from motile Aeromonas septicemia disease Antimicrobial resistance prevalence of Aeromonas hydrophila isolates from motile Aeromonas septicemia disease. In Science, IOP Conference Series: Earth and Environmental 137:012076. https://doi.org/10.1088/1755-1315/137/1/012076

Larsen AM, Mohammed HH, Arias CR (2014) Characterization of the gut microbiota of three commercially valuable warmwater fish species. J Appl Microbiol 116(6):1396–1404. https://doi.org/10.1111/jam.12475 PubMed DOI

Liu R, Lian Z, Hu X, Lü A, Sun J, Chen C, Liu X, Song Y, Yiksung Y (2019) First report of Vibrio vulnificus infection in grass carp Ctenopharyngodon idellus in China. Aquaculture 499:283–289. https://doi.org/10.1016/j.aquaculture.2018.09.051 DOI

Noga EJ (2010) Fish disease: diagnosis and treatment, 2nd edn. Willey, Iowa, pp 49–55 DOI

Oliveira RV, Peixoto PG, Ribeiro DDC et al (2014) Klebsiella pneumoniae as a main cause of infection in nishikigoi Cyprinus carpio (carp) by inadequate handling. Braz J Vet Parasitol 7(2):86–88

Pagniez H, Berche P (2005) Les infections à Shewanella, un pathogene opportuniste émergent. Med Mal Infect 35(4):186–191. https://doi.org/10.1016/j.medmal.2005.03.008 PubMed DOI

Parker JL, Shaw JG (2011) Aeromonas spp. clinical microbiology and disease. J Inf Secur 62(2):109–118. https://doi.org/10.1016/j.jinf.2010.12.003 DOI

Pękala A, Kozińska A, Paździor E, Głowacka H (2015) Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish. J Fish Dis 38(3):283–293. https://doi.org/10.1111/jfd.12231 PubMed DOI

Pérez-Sancho M, Cerdá I, Fernández-Bravo A, Domínguez L, Figueras MJ, Fernández-Garayzábal JF, Vela AI (2018) Limited performance of MALDI-TOF for identification of fish Aeromonas isolates at species level. J Fish Dis 41(10):1485–1493. https://doi.org/10.1111/jfd.12837 PubMed DOI

Persson S, Al-Shuweli S, Yapici S, Jensen JN, Olsen KE (2015) Identification of clinical Aeromonas species by rpoB and gyrB sequencing and development of a multiplex PCR method for detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media. J Clin Microbiol 53(2):653–656. https://doi.org/10.1128/JCM.01963-14 PubMed DOI PMC

Piamsomboon P, Jaresitthikunchai J, Hung TQ, Roytrakul S, Wongtavatchai J (2020) Identification of bacterial pathogens in cultured fish with a custom peptide database constructed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). BMC Vet Res 16:52. https://doi.org/10.1186/s12917-020-2274-1 PubMed DOI PMC

Roberts HE, Palmeiro B, Weber ES (2009) Bacterial and parasitic diseases of pet fish. Vet Clin North Am Exot Anim Pract 12(3):609–638. https://doi.org/10.1016/j.cvex.2009.06.010 PubMed DOI

Roubach R, Gomes LC, Leão Fonseca FA et al (2005) Eugenol as an efficacious anaesthetic for tambaqui, Colossoma macropomum (Cuvier). Aquac Res 36(11):1056–1061. https://doi.org/10.1111/j.1365-2109.2005.01319.x DOI

Russo R, Mitchell H, Yanong RPE (2006) Characterization of Streptococcus iniae isolated from ornamental cyprinid fish and development of challenge models. Aquaculture 256(1–4):105–110. https://doi.org/10.1016/j.aquaculture.2006.02.046 DOI

Saeed M, Alamoudi M, Al-Harbi A (1987) A Pseudomonas associated with disease in cultured rabbitfish Siganus rivulatus in the Red Sea. Dis Aquat Org 3:177–180. https://doi.org/10.3354/dao003177 DOI

Sebastião FA (2015) Validação de técnicas moleculares para o diagnóstico de bactérias em peixes, visando redução de tempo e custo. Tese, Universidade Estadual Paulista Júlio Mesquisa Filho

Sharifuzzaman SM, Rahman H, Austin DA, Austin B (2018) Properties of probiotics Kocuria SM1 and Rhodococcus SM2 isolated from fish guts. Probiotics Antimicrob Proteins 10(3):534–542. https://doi.org/10.1007/s12602-017-9290-x PubMed DOI

Silva ERDFS, Castro V, Prianti MG et al (2017) Occurrence of antibodies against Leptospira spp in dogs from Teresina, Piauí, Brazil. Braz J Vet Res Anim Sci 54(1):88. https://doi.org/10.11606/issn.1678-4456.bjvras.2017.110588 DOI

Sreedharan K, Philip R, Singh ISB (2011) Isolation and characterization of virulent Aeromonas veronii from ascitic fluid of oscar Astronotus ocellatus showing signs of infectious dropsy. Dis Aquat Org 94(1):29–39. https://doi.org/10.3354/dao02304 DOI

Steinhagen D, Katharina H, Ellmer B et al (1998) Goussia carpelli (Protozoa: Coccidia) infection in stressed and immunosuppressed commom carp Cyprinus carpio. Dis Aquat Org 34(3):199–204 DOI

Thanigaive S, Vijayakumar S, Gopinath S et al (2015) In vivo and in vitro antimicrobial activity of Azadirachta indica (Lin) against Citrobacter freundii isolated from naturally infected Tilapia (Oreochromis mossambicus). Aquaculture 437:252–255. https://doi.org/10.1016/j.aquaculture.2014.12.008 DOI

Topić Popović N, Kazazić SP, Strunjak-Perović I et al (2017) Differentiation of environmental aquatic bacterial isolates by MALDI-TOF MS. Environ Res 152:7–16. https://doi.org/10.1016/j.envres.2016.09.020 DOI

Trust TJ, Whitby JL (1976) Antibiotic resistance of bacteria in water containing ornamental fish. Antimicrob Agents Chemother 10(4):598–603 DOI

Vávrová A, Balážová T, Sedláček I, Tvrzová L, Šedo O (2015) Evaluation of the MALDI-TOF MS profiling for identification of newly described Aeromonas spp. Folia Microbiol (Praha) 60(5):375–383. https://doi.org/10.1007/s12223-014-0369-4 DOI

Walczak N, Puk K, Guz L (2017) Bacterial flora associated with diseased freshwater ornamental fish. J Vet Res 61(4):445–449. https://doi.org/10.1515/jvetres-2017-0070 PubMed DOI PMC

Wang E, Yuan Z, Wang K, Gao D, Liu Z, Liles MR (2019) Consumption of florfenicol-medicated feed alters the composition of the channel catfish intestinal microbiota including enriching the relative abundance of opportunistic pathogens. Aquaculture 501:111–118. https://doi.org/10.1016/j.aquaculture.2018.11.019 DOI

Weir M, Rajić A, Dutil L et al (2012) Zoonotic bacteria, antimicrobial use and antimicrobial resistance in ornamental fish: a systematic review of the existing research and survey of aquaculture-allied professionals. Epidemiol Infect 140(2):192–206. https://doi.org/10.1017/S0950268811001798 PubMed DOI

Whittington RJ, Chong R (2007) Global trade in ornamental fish from an Australian perspective: the case for revised import risk analysis and management strategies. Prev Vet Med 81(1–3):92–116. https://doi.org/10.1016/j.prevetmed.2007.04.007 PubMed DOI

Wu TS, Chiu CH, Yang CH, Leu HS, Huang CT, Chen YC, Wu TL, Chang PY, Su LH, Kuo AJ, Chia JH, Lu CC, Lai HC (2012) Fish tank granuloma caused by Mycobacterium marinum. PLoS One 7(7):e41296. https://doi.org/10.1371/journal.pone.0041296 PubMed DOI PMC

Wu CJ, Ko WC, Lee NY, Su SL, Li CW, Li MC, Chen YW, Su YC, Shu CY, Lin YT, Chen PL (2019) Isolates from fish and patients in Tainan City, Taiwan: genotypic and phenotypic characteristics. Appl Environ Microbiol 85(21):e01360–e01319. https://doi.org/10.1128/AEM.01360-19 PubMed DOI PMC

Yan L, Pei X, Zhang X, Guan W, Chui H, Jia H, Ma G, Yang S, Li Y, Li N, Yang D (2019) Occurrence of four pathogenic Vibrios in Chinese freshwater fish farms in 2016. Food Control 95:85–89. https://doi.org/10.1016/j.foodcont.2018.07.043 DOI

Yucel N, Aslim B, Beyatli Y (2005) Prevalence and resistance to antibiotics for Aeromonas species isolated from retail fish in Turkey. J Food Qual 28:313–324. https://doi.org/10.1111/j.1745-4557.2005.00037.x DOI

Zhang D, Xu DH, Shoemaker C (2016) Experimental induction of motile Aeromonas septicemia in channel catfish (Ictalurus punctatus) by waterborne challenge with virulent Aeromonas hydrophila. Aquac Rep 3:18–23. https://doi.org/10.1016/j.aqrep.2015.11.003 DOI