Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate.

The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require 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 in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.

Effect of storage temperature on survival and growth of foodborne pathogens on whole, damaged, and internally inoculated jalapeños (Capsicum annuum var. annuum).

There is a lack of general knowledge regarding the behavior of foodborne pathogenic bacteria associated with jalapeño peppers. The survival and growth behaviors of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica on the interior and exterior of jalapeño peppers were determined under different storage conditions. Jalapeños were inoculated with a five-strain cocktail of L. monocytogenes, E. coli O157:H7, or S. enterica on the intact external surface, injured external surface, or intact internal cavity of jalapeño peppers and held at 7 or 12°C for a period of 14 days. Populations of each pathogen were determined at 0, 1, 2, 5, 7 10, and 14 days throughout storage. The uninjured, intact external surface of jalapeño peppers did not support growth of the pathogens tested under both storage conditions, with the exception of L. monocytogenes at 12°C. Populations of E. coli and S. enterica declined on the external injured surface of peppers at 7°C, but populations of L. monocytogenes remained consistent throughout the length of storage. At 12°C, L. monocytogenes and S. enterica populations increased throughout storage, and E. coli populations remained unchanged on injured surfaces. The uninjured internal cavity of the jalapeño supported growth of all pathogens at 12°C. Overall, L. monocytogenes was the microorganism most capable of growth and survival in association with jalapeño peppers for the scenarios tested. Results emphasize the importance of jalapeño pepper quality and proper storage conditions in preventing or reducing pathogen survival and growth.

Label-free detection of multiple bacterial pathogens using light-scattering sensor.

Technologies for rapid detection and classification of bacterial pathogens are crucial for securing the food supply. This report describes a light-scattering sensor capable of real-time detection and identification of colonies of multiple pathogens without the need for a labeling reagent or biochemical processing. Bacterial colonies consisting of the progeny of a single parent cell scatter light at 635 nm to produce unique forward-scatter signatures. Zernike moment invariants and Haralick descriptors aid in feature extraction and construction of the scatter-signature image library. The method is able to distinguish bacterial cultures at the genus and species level for Listeria, Staphylococcus, Salmonella, Vibrio, and Escherichia with an accuracy of 90-99% for samples derived from food or experimentally infected animal. Varied amounts of exopolysaccharide produced by the bacteria causes changes in phase modulation distributions, resulting in strikingly different scatter signatures. With the aid of a robust database the method can potentially detect and identify any bacteria colony essentially instantaneously. Unlike other methods, it does not destroy the sample, but leaves it intact for other confirmatory testing, if needed, for forensic or outbreak investigations.

Analysis of time-resolved scattering from macroscale bacterial colonies.

We investigate the relationship of incubation time and forward-scattering signature for bacterial colonies grown on solid nutrient surfaces. The aim of this research is to understand the colony growth characteristics and the corresponding evolution of the scattering patterns for a variety of pathogenic bacteria relevant to food safety. In particular, we characterized time-varying macroscopic and microscopic morphological properties of the growing colonies and modeled their optical properties in terms of two-dimensional (2-D) amplitude and phase modulation distributions. These distributions, in turn, serve as input to scalar diffraction theory, which is, in turn, used to predict forward-scattering signatures. For the present work, three different species of Listeria were considered: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The baseline experiments involved the growth of cultures on brain heart infusion (BHI) agar and the capture of scatter images every 6 h over a total incubation period of 42 h. The micro- and macroscopic morphologies of the colonies were studied by phase contrast microscopy. Growth curves, represented by colony diameter as a function of time, were compared with the measured time-evolution of the scattering signatures.

Biophysical modeling of forward scattering from bacterial colonies using scalar diffraction theory.

A model for forward scattering from bacterial colonies is presented. The colonies of interest consist of approximately 10(12) - 10(13) individual bacteria densely packed in a configuration several millimeters in diameter and approximately 0.1-0.2 mm in thickness. The model is based on scalar diffraction theory and accounts for amplitude and phase modulation created by three macroscopic properties of the colonies: phase modulation due to the surface topography, phase modulation due to the radial structure observed from some strains and species, and diffraction from the outline of the colony. Phase contrast and confocal microscopy were performed to provide quantitative information on the shape and internal structure of the colonies. The computed results showed excellent agreement with the experimental scattering data for three different Listeria species: Listeria innocua, Listeria ivanovii, and Listeria monocytogenes. The results provide a physical explanation for the unique and distinctive scattering signatures produced by colonies of closely related Listeria species and support the efficacy of forward scattering for rapid detection and classification of pathogens without tagging.

Prevalence of antibodies reactive to pathogenic and nonpathogenic bacteria in preimmune serum of New Zealand white rabbits.

Polyclonal antibodies are typically produced in rabbits. The rabbit's health plays an important role in the quality of antibodies produced. Therefore, recommendations have been made by organizations on which bacteria and how frequently to test rabbit colonies. Since it is well known that rabbits may contain cross-reactive antibodies in their preimmune serum, it is common to test the rabbits for reactivity prior to immunization. Here preimmune sera from 19 different rabbits were tested with ELISA against 27 pathogenic and nonpathogenic bacterial cultures. ELISA results showed that Salmonella enterica serovar Typhimurium and Bacillus cereus AS4-12 had the highest average absorbance values (0.60 and 0.54, respectively) and the most preimmune serum samples testing positive was 17. Pseudomonas putrefaciens and B. subtilis had the lowest absorbance values (< 0.1) and did not test positive in any of the preimmune serum samples. Fourteen of the 27 cultures showed positive reactions with 50% or more of the preimmune serum samples tested. Fifty-three percent of the rabbit preimmune sera showed positive reactions with 10 or more bacterial cultures. In Western blot analyses, selected serum samples showing the highest ELISA values reacted with bands in the 97, 36, and 29 kDa regions or with bands in the 63 kDa and 32 kDa regions. Data suggest that the presence of cross-reactive antibodies in the preimmune serum is a common problem amongst the samples tested. Extensive preimmune serum testing should be implemented when polyclonal antibodies are intended for diagnostic testing.