title: 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 (ΔinlA strain), and an accessory secretory protein (ΔsecA2 strain). Here we show that the scatter patterns of lap mutant, ΔinlA, and ΔsecA2 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 ΔinlA 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.
  
  
  
  In 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.
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Atul K
authors.identifier: http://orcid.org/0000-0001-7343-3577
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Lena
authors.initials: L
authors.lastname: Leprun
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Rishi
authors.initials: R
authors.lastname: Drolia
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Xingjian
authors.initials: X
authors.lastname: Bai
authors.affiliation: School of Mechanical Engineering, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Huisung
authors.initials: H
authors.lastname: Kim
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Amornrat
authors.initials: A
authors.lastname: Aroonnual
authors.affiliation: School of Mechanical Engineering, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Euiwon
authors.initials: E
authors.lastname: Bae
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA.|Ivy Tech Community College, Lafayette, Indiana, USA.
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Mishra
authors.affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USA bhunia@purdue.edu.|Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Arun K
authors.identifier: http://orcid.org/0000-0003-3640-1554
authors.initials: AK
authors.lastname: Bhunia
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods*; D055592:Biophysical Phenomena; D007834:Lasers; D008089:Listeria monocytogenes / Q000737:chemistry / Q000145:classification*; D008565:Membrane Proteins / Q000032:analysis / Q000172:deficiency*; D013499:Surface Properties; D037521:Virulence Factors / Q000032:analysis / Q000172:deficiency*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D037521:Virulence Factors