A rapid bioassay for bactericides against the catfish pathogens Edwardsiella ictaluri and Flavobacterium columnare

Abstract

The most common bacterial diseases in pond-raised channel cat¢sh Ictalurus punctatus (Ra¢nesque) are enteric septicemia of cat¢sh and columnaris, caused by Edwardsiella ictaluri and Flavobacterium columnare respectively. Medicated feed containing antibiotics is one management approach that cat¢sh producers use in the treatment of bacterial diseases. However, the future use of all types of medicated feed in cat¢sh aquaculture is uncertain. To discover e¡ective alternatives to antibiotics, a rapid 96-well microplate bioassay utilizing E. ictaluri and F. columnare to evaluate natural compounds and extracts was developed. In this bioassay, bacterial growth is determined by absorbance measurements of microplate wells after 24 h incubation and then con¢rmed by detecting cell viability after the addition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide with additional incubation for 24 h. The minimum inhibitory concentration , minimum bactericidal concentration and 50% inhibition concentration (IC50) are determined by graphing the absorbance data. The 24 h IC50 results of test compounds are compared with the 24 h IC50 results of the drug controls oxytetracycline and £orfenicol. Among the antibiotics evaluated, doxycycline and tetracycline appear more e¡ective against E. ictaluri and F. columnare than either drug control. This bioassay is rapid, reproducible and economical for evaluating a large number of compounds and extracts.

Antibacterial Activity of Acylglucinol Derivatives against Flavobacterium columnare

“…Prior to bioassay, single colony of F. columnare was used to prepare the assay culture material by culturing in 75 mL of MS broth (24 h) at 29˚C ± 1˚C at 150 rpm on a rotary shaker (model C24KC; New Brunswick Scientific, Edison, New Jersey, USA). After overnight incubation, a 0.5 McFarland standard of F. columnare assay material was prepared by transferring cells from the broth culture to fresh MS broth [2].…”

“…Pure compounds (>98% pure) were evaluated for antibacterial activity using a rapid 96-well microplate bioassay and following the procedures of Schrader and Harries [2]. Florfenicol was included as a positive drug control.…”

“…In order to determine the 24-h 50% inhibitory concentration (IC 50 ) and minimum inhibitory concentration (MIC), sterile 96-well polystyrene microplates (Corning Costar Corp., Acton, Massachusetts, USA) with flat-bottom wells were used. Initially, reconstituted test compounds or drug control were aspirated separately into individual microplate wells (10 µL/well), and solvent was allowed to completely evaporate at room temperature before 0.5 MacFarl and bacterial culture (prepared as described by Schrader and Harries The means and standard errors of absorbance measurements were calculated, graphed, and compared to controls to determine the 24-h IC 50 and MIC of each test compound [2]. The 24-h IC 50 and MIC results were divided by the respective 24-h IC 50 and MIC results (in ppm) obtained for the positive control, to determine the relative-to-drug-control florfenicol (RDCF) values.…”

“…Direct economic losses to catfish producers are likely millions of dollars annually from single or mixed infections involving F. columnare [2]. The Gram-negative, rod-shaped bacterium Flavobacterium columnare is the cause of columnaris disease, and two genomovars of F. columnare have been identified, with genomovar II isolates attributed as being more pathogenic towards channel catfish [3].…”

“…As part of our continuing effort to discover environmentally benign compounds for the control of columnaris disease, acyl derivatives of phloroglucinol were synthesized and tested against F. columnare in a rapid bioassay developed by Schrader and Harries [2]. As a result of our investigation, we were able to identify several potent derivatives with activities similar to the drug control florfenicol.…”