Submitted to: Microbial Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/11/2012
Publication Date: 5/1/2012
Citation: Huff, K., Aroonnual, A., Bae, E., Banada, P., Rajwa, B., Rajwa, B., Hirleman, E.D., Robinson, J.P., Richards, G.P., Bhunia, A. 2012. Light scattering sensor for real-time identification of Vibrio parahaemolyticus, V. vulnificus and V. cholera colonies on solid agar plates. Microbial Biotechnology. DOI: 10.1111/j.1751-7915.2012.00349.x. Interpretive Summary: Vibrio species are increasingly becoming a major concern as seafood-associated illness occurs at increasing frequency worldwide. Although there is a need to detect vibrios in the shortest time possible, current methods are complex and time-consuming. This paper describes a procedure known as BARDOT (BActerial Rapid Detection using Optical light scattering Technology) which allows three dangerous vibrios (Vibrio cholera, V. vulnificus, and V. parahaemolyticus) and related species to be identified from colonies growing on agar plates. Colonies on the agar plates are illuminated by a laser light and colonies from different species of Vibrio produce a unique light scattering signature, which is consistent from colony to colony. A computer analyzes the scatter patterns and makes an identification based on pattern recognition software. In this manner, Vibrio colonies on agar plates were differentiated and classified with an accuracy of over 99% in 1-2 min, without compromising or destroying the integrity of the colonies, which may be used for further confirmation. Light scattering signatures were unaffected for bacteria that were subjected to stress including salt imbalance, acid, heat, and cells which recovered from a dormant state. Furthermore, the light scattering sensor successfully detected and identified Vibrio species from spiked raw oyster and water samples and the identity of each positive colony was confirmed. This technique provides a rapid, simple, and inexpensive tool for monitoring large numbers of potentially dangerous bacteria in a short period of time.
Technical Abstract: The three most common pathogenic species of Vibrio, V. cholerae, V. parahemolyticus and V. vulnificus, are of major concern as water- and food-borne pathogens because of an increasing incidence of water and seafood related outbreaks and illnesses worldwide. Current methods are time-consuming and require complex biochemical and molecular confirmation. A novel label-free forward-light scattering sensor was developed to detect and identify colonies of these three pathogens in real-time in the presence of other vibrios and/or in inoculated food or water samples. Vibrio colonies grown on agar plates were illuminated by 635-nm laser beam and scatter image signatures were acquired using a CCD camera in an automated BARDOT (BActerial Rapid Detection using Optical light scattering Technology) system. Each species of Vibrio produced a unique light scattering signature, which is consistent from colony to colony. Subsequently a pattern recognition system analyzing the collected light-scatter information provided classification with accuracy of over 99% in 1-2 minutes. The light scattering signatures of colonies were unaffected by subjecting the bacteria to physiological stress, including osmotic imbalance, acid, heat, as well as recovery from a viable but non-culturable state. Furthermore, the light scattering sensor successfully detected and identified V. cholerae, V. parahemolyticus and V. vulnificus present in spiked raw oyster or water samples and even in the presence of other vibrios in 12-18 h indicating the suitability of the light scattering sensor as a powerful identification and screening tool for pathogens on conventional agar plates.