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

Abstract

In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid detection using optical scattering technology) could be used to directly screen colonies of Listeria monocytogenes, a model pathogen, with mutations in several known virulence genes, including the genes encoding Listeria adhesion protein (LAP; lap mutant), internalin A (⌬inlA strain), and an accessory secretory protein (⌬secA2 strain). Here we show that the scatter patterns of lap mutant, ⌬inlA, and ⌬secA2 colonies were markedly different from that of the wild type (WT), with >95% positive predictive values (PPVs), whereas for the complemented mutant strains, scatter patterns were restored to that of the WT. The scatter image library successfully distinguished the lap mutant and ⌬inlA mutant strains from the WT in mixed-culture experiments, including a coinfection study using the Caco-2 cell line. Among the biophysical parameters examined, the colony height and optical density did not reveal any discernible differences between the mutant and WT strains. We also found that differential LAP expression in L. monocytogenes serotype 4b strains also affected the scatter patterns of the colonies. The results from this study suggest that BARDOT can be used to screen and enumerate mutant strains separately from the WT based on differential colony scatter patterns. IMPORTANCEIn studies of microbial pathogenesis, virulence-encoding genes are routinely disrupted by deletion or insertion to create mutant strains. Screening of mutant strains is an arduous process involving plating on selective growth media, replica plating, colony hybridization, DNA isolation, and PCR or immunoassays. We applied a noninvasive laser scatterometer to differentiate mutant bacterial colonies from WT colonies based on forward optical scatter patterns. This study demonstrates that BARDOT can be used as a novel, label-free, real-time tool to aid researchers in screening virulence gene-associated mutant colonies during microbial pathogenesis, coinfection, and genetic manipulation studies.

Tunicamycin Mediated Inhibition of Wall Teichoic Acid Affects Staphylococcus aureus and Listeria monocytogenes Cell Morphology, Biofilm Formation and Virulence

“…Plates were incubated at 37°C for 17–27 h or until the colony diameter reached 1.1 ± 0.2 mm. Colony images were acquired under a light microscope (100x magnification) (Leica) and colony scatter images of each pathogen, at least 40 colonies were collected using an automated BARDOT (BActerial Rapid Detection using Optical scattering Technology) machine ( Singh et al, 2014 , 2015 , 2016 ).…”

Simultaneous detection ofSalmonella enterica,Escherichia coliandListeria monocytogenesin food using a light scattering sensor

“…The low‐power (1 mW mm −2 ) red diode laser (635 nm) used in BARDOT does not affect bacterial viability; therefore, the live cultures obtained from test samples can be used for other experimentation such as to monitor the effect of sanitizers for controlling the pathogens in the food processing/production plants, antibiotic sensitivity testing, pathogenicity analysis or other molecular characterization (Bhunia ; Singh et al . ).…”

Petri-plate, bacteria, and laser optical scattering sensor

“…Hence, BARDOT has been considered a non-invasive, non-destructive, and reagent-free detection platform for pathogens of food and clinical relevance. In addition, BARDOT showed utility in differentiating mutant strains deficient in virulence-gene in L. monocytogenes ( Singh et al., 2016 ) or antibiotics-induced stress response by bacterial pathogens ( Singh et al., 2015a ; Zhu et al., 2018 ). The optical forward scattering technique was also evaluated for application in clinical microbiology for the detection of colonies of E. coli , S. aureus , Proteus mirabilis , Yersinia enterocolitica , and Salmonella Typhimurium in an automated pathogen identification platform with the variable as well as fixed incubation time ( Minoni et al., 2015 ).…”