Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/31/2005
Publication Date: 7/7/2005
Citation: Richards, G.P., Watson, M.A., Parveen, S.A. 2005. Development of a simple and rapid fluorogenic procedure for the identification of vibrionaceae family members. Applied and Environmental Microbiology. 71:3524-3527. Interpretive Summary: The Vibrio family consists of a wide variety of bacteria that cause life-threatening diseases in humans and fish. We developed a simple and rapid procedure to detect vibrios isolated from seawater, shellfish, sewage, and clinical samples. This method is known as the colony overlay procedure for peptidases (COPP) and is based on the detection of an enzyme that we recently discovered in all Vibrio family members tested to date. The COPP technique requires the overnight growth of bacterial colonies on agar plates followed by a 10-min overlay of the plate with a membrane containing a special fluorescent indicator. Vibrio colonies that come in contact with the indicator will produce bright fluorescent spots on the membrane and these spots can be counted to determine the levels of Vibrio present in shellfish, seawater, sewage, or in clinical isolates. The spots may be viewed under black light and photographed. Identification of isolates revealed that 93.5% of the fluorescent spots were caused by Vibrio family members. This procedure provides for the rapid, simple, and inexpensive identification of vibrios and may be valuable in clinical, environmental, and food-testing laboratories. Since fish are highly susceptible to infection by vibrios, the COPP assay may be particularly valuable for monitoring water quality and fish diseases in the aquaculture industry.
Technical Abstract: We describe a rapid, fluorogenic procedure to detect Vibrionaceae family members isolated from seawater, shellfish, sewage, and clinical samples. The assay, referred to as the colony overlay procedure for peptidases (COPP), is based on a recently identified lysyl aminopeptidase activity that is associated with phosphoglucose isomerase produced by members of the Vibrionaceae family. The COPP assay requires overnight growth of bacterial colonies on agar plates followed by a 10-min overlay of the colonies with cellulose acetate membranes containing a synthetic substrate. Membranes are observed for fluorescent foci under ultraviolet illumination. Fluorescent foci were produced by all Vibrionaceae tested including Vibrio vulnificus, V. cholerae O1 and O139, V. parahaemolyticus, Aeromonas hydrophila, A. veronii, Plesiomonas shigelloides, and six other Vibrio spp. Fluorescence was not produced on overlays of non-Vibrionaceae pathogens. Field samples of seawater and oysters were assayed and biochemical identifications were attempted on 170 colonies of which 120 produced fluorescent foci, 93 were identified, and 87 (93.5%) were Vibrionaceae, principally V. vulnificus, V. parahaemolyticus, A. hydrophila, Photobacterium damsiella, and Sherwanella putrafaciens. Only 13 of 47 non-fluorescent isolates could be identified and none were Vibrionaceae. No fluorescent foci were present on overlays of bacteria isolated from soil; however, ~20% of sewage isolates were A. hydrophila. The COPP procedure provides for the rapid, simple, and inexpensive identification and quantitation of Vibrionaceae and may be particularly valuable in clinical, environmental, and food-testing laboratories and for monitoring water quality and fish diseases in the aquaculture industry.