J. Med. Microbiol. - Vol. 35 (1991), 264-269 0 1991 The Pathological Society of Great Britain and Ireland Plasmid associated virulence properties of environmental isolates of Aerornonas h ydrophila J. J. BORREGO, M. A. MORIflIGO, E. MARTINEZ-MANZANARES, M. BOSCA, DOLORES CASTRO, J. L. BARJA" and ALICIA E. TORANZ0"t Department of Microbiology, Faculty of Sciences, University of Malaga, 2907 1 - Malaga and Department of Microbiologyand Parasitology, Faculty of Biology, University of Santiago, 15706- Santiago de Compostela,Spain Summary. The plasmid profiles, and their association with antimicrobial resistance, of 60 strains of Aeromonas hydrophila isolated from fish, shellfish and water were investigated. Only two strains were susceptibleto all the antimicrobial agents tested ; the highest incidences of resistance were to tetracycline (93.7%), prystanamycin (93.379, ampicillin (9 1 -7%) and cephalothin (91.7%). Forty strains harboured one or more plasmids and the plasmid profile most frequently detected (15%) was the association of three small plasmids of 4-2, 3.2 and 2.8 Mda. Curing experiments indicated that the plasmid-free derivative strains simultane- ously lost their resistance determinants to tobramycin, neomycin, gentamicin and kanamycin. More than 90% of the strains tested produced siderophores and displayed haemolytic activity. However, the relationship between these virulence characters and the presence of plasmids was different; in 74.5% of the strains there was siderophore production and plasmids were detectable, whereas only 60%of the strains simultaneously possessed plasmids and haemolytic activity. Introduction Aeromonas hydrophila causes several diseases among poecilotherms and warm blood animals, including haemorrhagic septicaemia, red sore, gastroenteritis and endocarditis. l4 In recent years, the increased development of the aquaculture of fish and shellfish has exacerbated the problems of infections of these animals caused by different pathogenic micro-orga- nisms. In addition, the extensive use of chemo- therapeutic agents in fish farms, as feed additives or administered into the water to prevent and treat fish diseases, has resulted in an increase of drug-resistant bacteria.' Several bacterial phenotypic properties, such as antimicrobial resistance or virulence factors, have been demonstrated to be plasmid encoded. The presence of plasmids in these potentially pathogenic micro-organisms may present a potential public health hazard, because they may be transferred from animals to man either directly of indire~tly.~? Several reports have shown that the presence of plasmids in clinically important bacteria increases their virulence. 7-1 Various virulence factors of Vibrio and Aeromonas spp. have been characterised, includ- ing extracellularproducts and protein molecules of the cell envelope.''-l4 One of the most important factors involved in the virulence of the pathogenic strains is Received 2 Nov. 1990; accepted 30 Jan. 1991. t Correspondence should be sent to Prof. A. E. Toranzo. the possession of an efficient iron-sequestering system mediated by sider~phores,"~ l6 which allows the bacteria to grow in the iron-limiting conditions imposed by the high-affinity iron-binding proteins present in organic fluids.g. 6r l7 In this study the antimocrobial resistance, the presence of haemolytic activity and the production of diffusible siderophores by A. hydrophila strains isolated from water and animals (shellfish and fish) were examined and the relationships between these viru- lence factors and the plasmid profiles of the strains were identified. Materials and methods Bacterial strains and culture conditions Sixty strains of A . hydrophiza isolated from animals (shellfishand fish) and water (freshwater and seawater) samples were used in this study. Stock cultures were maintained frozen at -20°C in Glycerol 15% v/v Tryptic Soy Broth (TSB; Difco) and, before electro- phoretic analysis, were grown in Brain-Heart Infusion Broth (BHIB; Difco) incubated at 28°C for 24-36 h. Antimicrobial susceptibility Resistance of the isolates to antimicrobial agents was determined by a disk diffusion method on Mueller- Hinton Agar (MHA; BioMerieux), with disks contain- ing (pg) : amikacin 30, ampicillin 10, carbenicillin 100, 264