Selection for Virulence in the Fish Pathogen Aeromonas Salmonicida, Using Coomassie Brilliant Blue Agar

Abstract

Coomassie Brilliant Blue Agar was used to quantify the frequency of the A-layer phenotype in different isolates of Aeromonas salmonicida. Hydrophilic, non-clumping isolates of A. salmonicida consisted predominantly of the A-layer minus phenotype. These bacteria were avirulent by intraperitoneal injection into susceptible brook trout (Salvelinus fontinalis) and could not be reisolated from infected fish. By contrast, hydrophobic, clumping isolates were predominantly of the A-layer positive phenotype, highly virulent in brook trout, and easily recovered from dead or moribund fish. A-layer positive and negative clones of A. salmonicida were derived by plating bacteria on Coomassie Blue Agar. The plating showed clearly that Coomassie Blue Agar could be used as a highly selective in vitro screening method to reclaim the virulence of certain isolates of A. salmonicida having a relatively low percentage of A-layer positive phenotypes.

INFECTION OF SEA LAMPREY WITH AN UNUSUAL STRAIN OFAEROMONAS SALMONICIDA

“…salmonicida isolates in pathogenesis for rate of mortality induction and cumulative percentage of mortality. The As-SL1 produced lighter colonies on CBBA, which may indicate an aberration within the outer cell envelope pertaining to the expression of the A-layer (Cipriano and Bertolini 1988), which is an important virulence factor of A. salmonicida (Daly et al 1996;Madetoja et al 2003). However, Johnson et al (1985) and Ward et al (1985) described virulent strains of A. salmonicida subsp.…”

“…Primary cultures were incubated at 22 C for up to 72 hr (AFS-FHS 2010). Bacterial growth was recorded, and individual colonies were subcultured onto TSA and Coomassie brilliant blue agar (CBBA; Cipriano and Bertolini 1988) for 48 hr at 22 C for biochemical and molecular analyses. Fish were also analyzed for other pathogenic bacteria, viruses, and parasites, according to the recommend protocols (OIE 2006;AFS-FHS 2010).…”

General and Specialized Media Routinely Employed for Primary Isolation of Bacterial Pathogens of Fishes

“…Although it may be readily isolated from infected fish or grown in laboratory cultures using TSA, BHIA, or other general media, TSA supplemented with 0.01% CBB (Udey, 1982;Cipriano and Bertolini, 1988) offers an excellent presumptive identification. Suspect blue colonies are visible within 48 hr, and single colonies can be selected for easy and quick characterization with a few simple additional tests.…”

“…It is not often that a bacterial species possesses a unique phenotype, so the goal is to select a character that a minimal number of bacteria will share. An example of this is Coomassie brilliant blue (CBB) medium (Udey, 1982;Cipriano and Bertolini, 1988), which is an effective differential medium for field and laboratory work with Aeromonas salmonicida. Coomassie brilliant blue in the medium stains the A-layer protein on the cell surface of A. salmonicida, resulting in blue colonies.…”

On the validity of setting breakpoint minimum inhibition concentrations at one quarter of the plasma concentration achieved following oral administration of oxytetracycline

“…Plates were examined for colonies with morphology, consistency and pigmen-tation typical of A. salmonicida (Bernoth, 1997). Randomly selected colonies were purified and tested for their colonial morphology on Coomassie Brilliant Blue Agar (CBBA), according to the method of Cipriano and Bertolini (1988) and for their reaction with A. salmonicida antisera (Bionor, Mono-As, BioNor Aqua, Skien, Norway). Minimum inhibitory concentration (MIC) of OTC against strains isolated in this work was established using the protocols of Alderman and Smith (2001) using Muller Hinton agar (Difco, Sparks, USA).…”