Laser Optical Sensor, a Label-Free On-Plate Salmonella enterica Colony Detection Tool

Abstract

We investigated the application capabilities of a laser optical sensor, BARDOT (bacterial rapid detection using optical scatter technology) to generate differentiating scatter patterns for the 20 most frequently reported serovars of Salmonella enterica. Initially, the study tested the classification ability of BARDOT by using six Salmonella serovars grown on brain heart infusion, brilliant green, xylose lysine deoxycholate, and xylose lysine tergitol 4 (XLT4) agar plates. Highly accurate discrimination (95.9%) was obtained by using scatter signatures collected from colonies grown on XLT4. Further verification used a total of 36 serovars (the top 20 plus 16) comprising 123 strains with classification precision levels of 88 to 100%. The similarities between the optical phenotypes of strains analyzed by BARDOT were in general agreement with the genotypes analyzed by pulsed-field gel electrophoresis (PFGE). BARDOT was evaluated for the real-time detection and identification of Salmonella colonies grown from inoculated (1.2 × 102 CFU/30 g) peanut butter, chicken breast, and spinach or from naturally contaminated meat. After a sequential enrichment in buffered peptone water and modified Rappaport Vassiliadis broth for 4 h each, followed by growth on XLT4 (~16 h), BARDOT detected S. Typhimurium with 84% accuracy in 24 h, returning results comparable to those of the USDA Food Safety and Inspection Service method, which requires ~72 h. BARDOT also detected Salmonella (90 to 100% accuracy) in the presence of background microbiota from naturally contaminated meat, verified by 16S rRNA sequencing and PFGE. Prolonged residence (28 days) of Salmonella in peanut butter did not affect the bacterial ability to form colonies with consistent optical phenotypes. This study shows BARDOT’s potential for nondestructive and high-throughput detection of Salmonella in food samples.

Virulence Gene-Associated Mutant Bacterial Colonies Generate Differentiating Two-Dimensional Laser Scatter Fingerprints

“…We observed that the scatter patterns of the WT and mutant strains were different on BHIA and LBA but were similar on different brands of BHIA. It has been reported in our previous studies that bacterial colony scatter patterns vary with the growth medium used (5,7,9,11). Thus, we expect that any mutation in the genes involved in the biochemical pathways for substrate utilization will also result in differential scatter patterns.…”

“…BARDOT proved to be useful for studying the streptomycin-induced stress response in Salmonella enterica serovar Typhimurium (12) and as a bioanalytical detection tool to validate the performance of a sample processing and enrichment device, i.e., PED (pathogen enrichment device) (13). BARDOT-generated colony scatter phenograms exhibited strong relationships with the genotypes of bacteria (5), suggesting that BARDOT can be used to screen mutant strains that are deficient in specific virulence genes. In the present study, we used Listeria monocytogenes as a model pathogen and used BARDOT to screen mutant strains that are deficient in specific virulence genes.…”

Salmonella in Shell Eggs: Mechanisms, Prevention and Detection

“…Over the last several decades, there have been significant developments in rapid Salmonella detection methods using multiple novel approaches reducing preenrichment or detection time (e.g., In-situ immuno-gold nanoparticle network ELISA biosensors, immunomagnetic separation, automated microfiltration system, pathogen enrichment device, laser optical sensor, modified media, isothermal amplication) [79][80][81][82][83][84][85][86][87][88][89]. (Figure 2).…”

“…One major recent development has been the reports of the use of an enzyme based approach coupled to a short enrichment and microfiltration steps for reducing time for sample preparation. This allowed Salmonella to be detected at very low levels (≤ 1 CFU/g) in different contaminated foods in 8 hours or less than one work shift [82][83][84]. The major concept is shown in Figure 3.…”

Instant scanner device for identifying wound infection utilizing Mie scatter spectra

“…Our technique uses back scatter spectra from a tissue surface, unlike Mie scatter imaging which uses forward scatter images through colonies to identify patterns 21 . Mie scatter imaging systems, often referred to as BARDOT systems, require bacteria to be cultured on before identification of bacterial species, as BARDOT systems are used to identify fully developed cultures on an optically transparent substrate 22,23,24 . The system designed here does not require the additional steps of culturing a wound sample to determine the presence or cause of bacterial infection.…”