Extracellular Protease, Extracellular Haemolysin, and Virulence in Aeromonas salmonicida

Abstract

We have examined the possibile relationships between extracellular protease and haemolysin and virulence in Aeromonas salmonicida, and the possible plasmid-encoded origin of these extracellular enzyme activities. Variants, isolated from different strains, showed simultaneous loss of both protease and haemolysin. The frequency of spontaneous loss from one strain (0.1–0.4%) as well as the frequency of induced loss from four strains (0.3–18%) treated with ethidium bromide suggested a plasmid-encoded origin for these enzymes. However, plasmid analyses showed no detectable loss of plasmids correlated with the loss of the extracellular activities. There was no change in the LD100 when fish were exposed to one virulent strain or its protease, haemolysin-negative variants indicating that these extracellular activities are not necessary for virulence or pathogenicity in the acute form of furunculosis. Furthermore, both protease, haemolysin-negative and protease, haemolysin-positive clones isolated from a second virulent strain treated with ethidium bromide were avirulent (LD50 increased >4 logs). Thus, attenuation of A. salmonicida can occur without detected loss of the extracellular enzymes, plasmids, or the A-layer.

Characterization of Aeromonas salmonicida mutants with low-level resistance to multiple antibiotics

“…Prt-mlr mutants (e.g., AsLAR-N1) were isolated from the AsLAR mutant as colonies grown on BHI agar plates containing 0.4 jig of nalidixic acid ml-' as described above and subsequently checked for antibiotic susceptibilities (34) and exoprotease (10).…”

“…To obtain protease-positive (Prt+) revertant strains, single colonies of the Prt-mlr mutants grown on BHI agar were inoculated into 250-ml Erlenmeyer flasks containing 50 ml of casein broth medium consisting of (liter-'): K2HPO4, 21 g; KH2PO4, 9 g; Casamino Acids (Difco Laboratories, Detroit, Mich.), 1 g; L-tryptophan, 0.03 g; glucose, 10 g; MgSO4 7H20, 0.05 g; and casein, 10 g. The flasks were incubated at 22°C and 150 rpm for .72 h. Samples of the cultures were plated on BHI agar containing a skim milkagar overlay and incubated at 22°C to detect Prt+ revertant colonies (10).…”

Multiple low-level antibiotic resistance in Aeromonas salmonicida

“…agar plates were incubated at 22°C for 24 h. BHI broths were incubated at 22°C for 24 h on a shaker (model G-26; New Brunswick Scientific Co., New Brunswick, N.J.) at 150 rpm. Mutants lacking exoprotease (caseinase) activity (Prt-) were isolated on skim milk overlay plates with BHI agar as a basal medium following subculture of lyophilized cultures or treatment ofthe cultures with ethidium bromide as previously described (9). Mutants isolated as antibiotic resistant were selected as colonies growing on BHI agar plates containing ampicillin (0.5 ,ug ml-'), chloramphenicol (1.5 jig ml-'), flumequine (0.1 ,ug ml-'), penicillin (4 ,ug ml-'), or rifampin (8 jig ml-').…”

“…The plates were surface inoculated from BHI broth cultures with approximately 2 x 108 cells as estimated by optical density. The antibiotic-resistant mutants were checked for exoprotease activity by plating on skim milk agar overlay plates (9). Mutants lacking the A layer (A-) were isolated following incubation of cultures at elevated temperature, as described by Ishiguro et al (14), and were checked for the presence of A layer by transmission electron microscopy (9).…”

“…f A-layer-negative strains isolated after incubation at elevated temperature (14). g Spontaneously occurring Prt-derivative of ATCC 14174 (9).…”