Virulence Gene-Associated Mutant Bacterial Colonies ...

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Virulence Gene-Associated Mutant Bacterial Colonies Generate Differentiating Two-Dimensional Laser Scatter Fingerprints Atul K. Singh,a Lena Leprun,a* Rishi Drolia,a Xingjian Bai,a Huisung Kim,b Amornrat Aroonnual,a* Euiwon Bae,b Krishna K. Mishra,a,c Arun K. Bhuniaa,d Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, USAa; School of Mechanical Engineering, Purdue University, West Lafayette, Indiana, USAb; Ivy Tech Community College, Lafayette, Indiana, USAc; Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USAd

ABSTRACT

IMPORTANCE

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.

A

laser scatterometer, designated BARDOT (bacterial rapid detection using optical scattering technology), operates on the principle of elastic light scattering. BARDOT uses a 635-nm reddiode laser (1 mW) beam shined onto the center of a bacterial colony on an agar plate, which generates a 2-dimensional luminous forward scatter fingerprint (1). Analyses of BARDOT images involve hundreds of independent parameters and features that are extracted from each scatter pattern to build a database of images for a specific microorganism (2–4). BARDOT has been used successfully for the detection of multiple bacterial pathogens, including Listeria spp. (2, 3), Salmonella enterica (5), Shiga-toxigenic Escherichia coli (6), Vibrio spp. (7), Bacillus spp. (8, 9), Campylobacter spp. (10), and several genera of the Enterobacteriaceae family (11). 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. Listeria monocytogenes is a Gram-positive, motile, rod-shaped,

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microaerophilic pathogenic bacterium that causes listeriosis. L. monocytogenes is ubiquitous in nature and is present in soil, water, and sewage as a saprophyte and in the intestines of cattle and sheep as an intracellular pathogen (14). Ready-to-eat meat and dairy products and fruits and vegetables act as carriers for this foodborne pathogen (15). L. monocytogenes is a major concern for immunocompromised individuals, including the elderly, neonates, and pregnant women and their fetuses (16). In the United States, listeriosis has the third highest fatality rate (19%) among the foodborne pathogens (17), and globally, the estimated perinatal infection rate is 20.7% (18).

Received 28 December 2015 Accepted 16 March 2016 Accepted manuscript posted online 18 March 2016 Citation Singh AK, Leprun L, Drolia R, Bai X, Kim H, Aroonnual A, Bae E, Mishra KK, Bhunia AK. 2016. Virulence gene-associated mutant bacterial colonies generate differentiating two-dimensional laser scatter fingerprints. Appl Environ Microbiol 82:3256 –3268. doi:10.1128/AEM.04129-15. Editor: J. L. Schottel, University of Minnesota Address correspondence to Arun K. Bhunia, [email protected]. * Present address: Lena Leprun, CROUS de Dijon, Dijon Cedex, France; Amornrat Aroonnual, Department of Tropical Nutrition & Food Science, Mahidol University, Bangkok, Thailand. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology

June 2016 Volume 82 Number 11

Downloaded from http://aem.asm.org/ on July 4, 2018 by guest

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.

Laser Scatterometer for Screening Bacterial Mutants

TABLE 1 Wild type, mutant, and complemented bacterial strains used in this study Species L. monocytogenes

F4244 F4244 KB208 (lap mutant) CKB208 (lap mutant lap⫹)

Relevant propertiesa

Source or reference s

AKB301 (⌬inlA) AKB302 (⌬inlA inlA⫹) AKB103 (⌬secA2) AKB104 (⌬secA2 secA2⫹) H4

WT, serovar 4b; DUP-1044; Em WT; Emr (10 ␮g/ml) lap insertion mutant in F4244; Emr (10 ␮g/ml) KB208 containing pCLAP-67 expressing lap in F4244; Emr (5 ␮g/ml) Cmr (5 ␮g/ml) inlA in-frame deletion in F4244 inlA complementation of AKB301 in F4244; Cmr (5 ␮g/ml) secA2 in-frame deletion in F4244 secA2 complementation of AKB104 in F4244; Emr (10 ␮g/ml) WT, serovar 4b; DUP-1042

10403S DP-L4342 (⌬secA2) F4262

WT, serovar 1/2a secA2 in-frame deletion in 10403S WT, serovar 4b; DUP-1051

F4248 F4248 (⌬secA2)

WT secA2 in-frame deletion mutant

CDC, Atlanta, GA Our collection 31 31 24 24 32 32 Blood of a 34-year-old patient, Sao Paulo, Brazil (62) D. Portnoy, UC-Berkeley (63) D. Portnoy, UC-Berkeley (29) CDC, listeriosis patient, cerebrospinal fluid/blood CDC, Atlanta, GA 28

a All strains were grown at 37°C, except for KB208, which was grown at 42°C. WT, wild type; Em, erythromycin; Cm, chloramphenicol. DUP numbers were obtained after comparing the ribopatterns with the RiboPrinter database (DuPont, Qualicon Inc.) for culture identification.

Upon consumption, L. monocytogenes penetrates the intestinal cell lining and reaches the mesenteric lymph nodes, liver, spleen, and brain to cause flu-like symptoms, headache, febrile gastroenteritis, and meningitis (19). In pregnant women, L. monocytogenes crosses the fetoplacental barrier to infect the fetus and may cause stillbirth, premature birth, and fetal sepsis. L. monocytogenes produces an array of virulence factors, such as adhesins and invasins, including internalin A (InlA), which functions in epithelial cell invasion during the intestinal and uteroplacental phases of infection (20, 21); internalin B (InlB) (22), which aids in the invasion of endothelial cells and colonization of the liver and spleen (23); and Listeria adhesion protein (LAP), which is responsible for adhesion to and translocation across Caco-2 cell monolayers (24). Additionally, hemolysin (Hly), phospholipase C (PLC), and actin polymerization (ActA) proteins are responsible for intracellular survival, cell-to-cell spread, and pathogenesis (25, 26). Many of the virulence proteins that are synthesized in the cytoplasm of the bacterium are translocated across the cell wall with the help of the novel secretory protein SecA2 to aid in the infection process (27–29). In studies of microbial pathogenesis, virulence-encoding genes are routinely disrupted by deletion or insertion to create mutant strains. The screening of mutant strains is a laborious process that involves plating on agar plates with selective agents (antibiotics and chromogens), replica plating, colony hybridization, DNA isolation, and PCR or immunoassays (30). Therefore, the primary aim of this study was to investigate whether BARDOT could be used to screen L. monocytogenes mutant colonies deficient in several key virulence factors, including LAP, InlA, and SecA2, directly on agar plates. MATERIALS AND METHODS Bacterial strains and culture conditions. The wild-type (WT) Listeria monocytogenes strain F4244 is an isolate from the cerebrospinal fluid of a listeriosis patient, and the culture was originally obtained from the Centers for Disease Control and Prevention (CDC). Mutant strains of F4244 with knockdown (KB208 [lap mutant] [31]) or knockouts (AKB301 [⌬inlA strain] [24] and AKB103 [⌬secA2 strain[ [32]) of virulence and


adhesion genes and mutant strains with complemented genes (CKB208 [lap mutant lap⫹] [31], AKB302 [⌬inlA inlA⫹] [24], and AKB103 [⌬secA2 secA2⫹] [32]) were constructed in our previous studies (Table 1). The WT and isogenic mutant strains were grown at either 37°C or 42°C at 130 rpm in an orbital shaker for 16 to 18 h. The Listeria adhesion protein knockdown and erythromycin-resistant KB208 strain (lap mutant) and the erythromycin-resistant WT strain (WT Emr) were grown in tryptic soy broth containing 0.6% yeast extract (TSBYE; Becton Dickinson, Franklin Lakes, NJ) supplemented with 10 ␮g/ml of erythromycin at 42°C and 37°C, respectively. In-frame deletion mutant strains of the ⌬inlA (AKB301) and ⌬secA2 (AKB103) strains were grown in TSBYE without any antibiotic at 37°C. The complemented strain CKB208 (lap mutant lap⫹) was grown at 37°C in TSBYE supplemented with 5 ␮g/ml each of erythromycin and chloramphenicol, the AKB302 (⌬inlA inlA⫹) strain was grown in TSBYE containing 5 ␮g/ml of chloramphenicol, and the AKB104 (⌬secA2 secA2⫹) strain was grown in TSBYE containing 10 ␮g/ml of erythromycin. For the light scattering analyses, the cultures were 10-fold serially diluted in 10 mM phosphate-buffered saline (PBS), pH 7.4, and appropriate dilutions were plated on brain heart infusion agar (BHIA) and LuriaBertani agar (LBA) plates containing appropriate antibiotics to obtain approximately 30 to 100 colonies per plate. The plates were incubated at 37°C or 42°C for 21 to 27 h to obtain a colony diameter of 1.0 ⫾ 0.2 mm to capture the scatter patterns by use of BARDOT. We also examined the scatter signatures of L. monocytogenes (WT) on BHIA procured from three suppliers (Becton Dickinson, Acumedia [Lansing, MI], and Teknova [Hollister, CA]) to monitor any potential effects of different brands on the colony scatter patterns. Laser scatterometer and image analyses. A machine that functions on biophysical principles, designated BARDOT, was invented and built at Purdue University by our group (1, 3, 33). BARDOT consists of the following three main parts: (i) a colony locator in which a back-illumination light with a diffuser aids in the localization of the colonies on the plate, (ii) a moving platform that moves the petri dish in the x and y directions, and (iii) a red-diode laser (635 nm; 1 mW; 1-mm diameter) and a complementary metal oxide semiconductor (CMOS) camera that illuminates and captures the image of each colony. The system provides a two-dimensional map for locating each colony on the plate based on the traveling salesman algorithm (33). Centering of the colonies is performed automatically by balancing the image intensity. The forward scatter patterns for colonies of 1.0 ⫾ 0.2 mm in diameter and with a roundness close to 0.7


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L. innocua

Strain

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study, ELISA was performed using the LAP-specific monoclonal antibody (MAb) Em10 (EM-H7) and the InlA-specific MAb 2D12, each at a 1:1,500 dilution, and an anti-mouse secondary antibody at a 1:2,000 dilution (Jackson ImmunoResearch Labs, West Grove, PA). Approximately 5 to 7 colonies identified by use of BARDOT from three replicate plates were selected using colony picks (Qualicon, Wilmington, DE), suspended directly in 100 ␮l of carbonate coating buffer (3.5 mM NaHCO3 and 1.5 mM Na2CO3) in the wells of a microtiter plate (4⫻ binding; Immulon), and incubated at 4°C for 24 h to perform the immunoassay. Colorimetric or fluorescence signals were generated after the addition of the chromogenic substrate o-phenylenediamine (OPD; Sigma-Aldrich) or the fluorescence substrate QuantaBlue (Thermo Scientific, Waltham, MA) and then measured at 450 nm or at 320 nm (excitation)/460 nm (emission) by use of a spectrophotometric plate reader (Benchmark; Bio-Rad) or a Gemini Fluormax plate reader (Molecular Devices, Sunnyvale, CA). At the same time, five colonies were also selected and resuspended in 100 ␮l PBS, and appropriate dilutions were plated on BHIA to count the number of cells per colony, which was 2.53 ⫻ 108 ⫾ 0.19 ⫻ 108 CFU. For coinfection study, Caco-2 cells (human colon adenocarcinoma cell line; ATCC, Manassas, VA) were infected with single (WT, lap mutant, or ⌬inlA strain) or mixed (WT and lap mutant or WT and ⌬inlA strain) cultures of L. monocytogenes. Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (with high glucose; HyClone, Logan, UT) with 10% (vol/vol) fetal bovine serum (FBS; Atlanta Biologicals, GA) at 37°C with 7% CO2 in a humidified cell culture incubator as described previously (38). Approximately 3.5 ⫻ 104 Caco-2 cells were seeded in 12-well tissue culture plates (TPP, Switzerland) to grow for 14 days or until 95% confluence was reached, and the cell culture medium was changed twice a week. Bacterial cultures were grown for 18 h and to an OD600 of ⬃1, equivalent to 8 ⫻ 108 ⫾ 0.02 ⫻ 108 CFU/ml, and further diluted for coinfection. Caco-2 cells (4 ⫻ 105 ⫾ 0.2 ⫻ 105) were infected with 1 ml of bacteria (4.1 ⫻ 106 ⫾ 0.1 ⫻ 106 CFU/ml) at a multiplicity of infection (MOI) of 10:1 for 1 h in serum-free DMEM, as described previously (39). For coinfection, the WT and mutant cultures were mixed at a 1:1 ratio (⬃2 ⫻ 106 CFU/ml of each strain). Wells were washed thrice with serum-free DMEM and lysed with 1 ml of sterile 0.1% Triton X-100, and the samples were serially diluted prior to plating on BHIA and BHIA supplemented with 10 ␮g/ml of erythromycin. Colonies on BHIA plates were scanned to acquire scatter patterns for differential enumeration of the WT and mutant strains as described for the mixed-culture study described above. The results were expressed as numbers of adhered bacterial colonies (CFU per milliliter) and as percentages of bacterial colonies adhered to Caco-2 cells. Scatter patterns of strains with variable virulence protein expression. To confirm the levels of LAP expression on the cell surface for the three L. monocytogenes serotype 4b strains (i.e., H4, F4244, and F4262), cell wall protein fractions were prepared (32), and at the same time the cultures were also plated on BHIA and LBA to capture the scatter patterns of colonies as described before. For protein analyses, 200 ml of each of the bacterial cultures (H4, F4244, and F4262) was grown in LB broth for 16 h at 37°C or until an OD600 of ⬃1.0. ELISA was performed as previously described (31). In brief, 10 ␮g of each of the cell wall protein fractions was separately resuspended in 1 ml of carbonate coating buffer, 100 ␮l was loaded per well (1 ␮g) in triplicate, and the plates were incubated at 4°C for 24 h. The LAP-specific primary MAb Em10 (1:2,000 dilution) and a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:4,000 dilution) were used in the immunoassay as described in the previous section. SDS-PAGE and Western blotting. Differential levels of LAP produced by L. monocytogenes F4244 (WT), F4262, H4, and KB208 (lap mutant) were quantified by Western blotting. The cell wall (CW) and intracellular (IC) protein fractions were extracted as described previously (32), and protein concentrations were estimated using a Pierce bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific). For Western blot-based validation of adhesion and virulence gene expression in the WT, mutant,




mm are captured by passing the laser beam from the top of the colonies and applying an average image capture time of 2 to 3 s/colony (33). A total of 60 scatter images per strain from two independent experiments with two technical replicates were acquired to train the scatter image libraries of the mutant and WT strains. Scatter patterns were analyzed by using a custom-developed pattern recognition algorithm (image classifier) that relies on machine-learning algorithms and training with scatter images of reference bacteria, and data are presented as classification accuracies or positive predictive values (PPVs) (34, 35). Various factors, including the rotation parameter invariant, are characterized by the Zernike moment (order of 8) and the textures that are used in feature extraction. The image classifier was used to extract and compute features from the scatter pattern in terms of the Zernike moment (ring and radial features) and Haralick texture (granularity/texture of the image) (35). This classifier generates similarity matrices (or cross-validation matrices) after analyses of the colony scatter pattern as numerical values that are used in computing PPVs and in the construction of the principal component analysis (PCA) chart. Time-resolved analyses and modeling of the light propagation through bacterial colonies have been performed to achieve a fundamental understanding of forward light scattering through a bacterial colony (36). Biophysical characteristics of WT and mutant colonies. The colony images were captured with a phase-contrast microscope. An integrated colony morphology analyzer (ICMA) (37), which is capable of simultaneously measuring three different optical modalities (i.e., spatial morphology, optical density [OD], and reflectance) of a single colony, was used to determine the height, transmittance, and reflectance of each colony. Because the OD of a single colony is not measurable with a spectrophotometer without destroying the colony integrity, the ODs of single colonies were computed indirectly on the agar plate with the ICMA, without deforming the colonies. The ICMA operates in a profile with a width of 1,100 ␮m and scans at intervals of 2 ␮m in both the x and y directions to capture the complete morphology and spatial OD map of a colony. The complete measurement of a single colony required approximately 15 to 20 min, and a cross-sectional measurement at the center region of a single colony required approximately 1.5 to 3 s. The colony profiles for a minimum of 15 colonies were captured for each strain, and average values were plotted. Analyses of scatter patterns of the WT, mutant, and complemented strains. The scatter patterns of the colonies of the individual mutant strains (i.e., lap mutant, ⌬inlA, and ⌬secA2 strains) on BHIA and LBA were acquired with BARDOT. Similarly, the scatter patterns of the colonies of the respective complemented strains (i.e., lap mutant lap⫹, ⌬inlA inlA⫹, and ⌬secA2 secA2⫹ strains) were also captured. The scatter patterns of the WT and WT Emr strains were also captured and were used as references to observe the differences in the scatter patterns of the mutant strains and the restoration of the scatter features of the complemented strains. The colony sizes and single-cell images of the WT, mutant, and complemented L. monocytogenes strains were also observed under a phase-contrast microscope with 10⫻ and 100⫻ objectives, respectively, with corresponding magnifications of ⫻100 and ⫻1,000. Most of the bacteria were imaged as doublets at the binary fission stage in the phasecontrast images. Mixed-culture (coculture and coinfection) experiment and enzymelinked immunosorbent assay (ELISA). To test the scatter pattern-based differentiation capability of BARDOT, two separate sets of mixed cultures (cocultures), i.e., the WT strain plus the ⌬inlA strain and the WT Emr strain plus the lap mutant strain, were plated on BHIA and BHIA supplemented with 10 ␮g/ml erythromycin, respectively. Cultures were grown for 16 to 18 h or until the OD at 600 nm (OD600) reached 1 (equivalent to 6.0 ⫻ 108 ⫾ 0.8 ⫻ 108 CFU/ml), and then approximately 30 to 50 CFU from the appropriate dilution of each strain were mixed and plated on BHIA. Scatter patterns were acquired and analyzed as described in the previous section. To validate the BARDOT-identified colonies in the mixed-culture


TABLE 2 Analysis of scatter patterns of colonies of wild-type and mutant strains of L. monocytogenes F4244 serotype 4b grown on BHIA and LBA Avg score (%) ⫾ SEMb Scatter image library and L. monocytogenes straina Sensitivity Specificity

NPV

PPV

BHIA library F4244 (WT) KB208 (lap mutant) AKB301 (⌬inlA) AKB103 (⌬secA2)

96.9 ⫾ 3.4 97.7 ⫾ 0.3 95.0 ⫾ 2.2 91.2 ⫾ 1.0

99.0 ⫾ 0.5 99.0 ⫾ 0.7 96.4 ⫾ 0.1 98.3 ⫾ 0.5

93.9 ⫾ 0.1 93.9 ⫾ 0.2 96.6 ⫾ 0.7 94.6 ⫾ 1.2

97.2 ⫾ 1.7 97.3 ⫾ 2.1 93.7 ⫾ 6.2 95.4 ⫾ 1.6

LBA library F4244 (WT) KB208 (lap mutant) AKB301 (⌬inlA) AKB103 (⌬secA2)

100 ⫾ 0 99.3 ⫾ 0.9 99.5 ⫾ 0.1 99.6 ⫾ 0.5

100 ⫾ 0 99.9 ⫾ 0.1 99.7 ⫾ 0.1 99.8 ⫾ 0.2

99.5 ⫾ 0.1 99.5 ⫾ 0.2 99.8 ⫾ 0.1 99.6 ⫾ 0.1

100 ⫾ 0 99.8 ⫾ 0.2 99.1 ⫾ 0.1 99.6 ⫾ 0.1

FIG 1 Scatter patterns and colony profiles of eight L. monocytogenes WT, mutant, and complemented strains. (a) Representative images of single cells

(magnification, ⫻1,000) and colonies (~1-mm diameter; magnification, ⫻100) of L. monocytogenes strains on brain heart infusion agar (BHIA) and Luria-Bertani agar (LBA) media. Bars, 2.5 ␮m. (b) Relative fold changes in the single-cell size of mutant strains of L. monocytogenes compared with the WT strain. The single cells were imaged with a phase-contrast microscope at a magnification of ⫻1,000, and the dimensions were measured with the ImageJ program. (c) Effects of temperature and erythromycin on scatter patterns of L. monocytogenes WT and WT Emr strains on BHIA. Images 1 and images 2 were acquired from two different experiments. See Table 1 for details on the strains.



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a Scatter image libraries consisted of a minimum of 60 scatter images of each strain obtained from two different experiments. BHIA, brain heart infusion agar; LBA, Luria-Bertani agar. b Scores were calculated by analyzing scatter patterns acquired in two independent experiments with two replicates (n ⫽ 4).

and complemented strains, all of the protein samples were prepared from whole-cell lysates, except for the ⌬secA2 clones (cell wall fraction). Equal amounts of proteins (11 ␮g) were separated in an SDS-PAGE gel (7.5% acrylamide) by using a Criterion cell system (Bio-Rad, Hercules, CA) and were then transferred to an Immobilon-P membrane (Millipore) by using a Criterion blotter (Bio-Rad). The membranes were blocked with 5% nonfat skim milk (NFSM) and dissolved in PBST (PBS plus 0.2% Tween 20) for 2 h at room temperature. The membranes were probed with the following primary antibodies at 4°C for 18 h in a blocking buffer (5% NFSM and PBS with 0.2% Tween 20): (i) LAP-specific MAb Em10 at a 1:1,000 dilution, (ii) InlA-specific MAb 2D12 at a 1:750 dilution, and (iii) N-acetylmuramidase (NamA)-specific MAb C11E9 at a 1:1,000 dilution (40). The membranes were stripped with Restore Plus Western blot stripping buffer (Thermo Scientific) before being probed with another primary antibody following the vendor’s instructions. HRP-conjugated antimouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch) were used at a 1:2,000 dilution for 1 to 1.5 h and washed thrice with PBST for 5 to 10 min at room temperature before development of the mem-

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RESULTS

Colony scatter patterns reveal differences between WT and mutant strains. To gain insights into the effect of genetic variability on colony morphotype, we used L. monocytogenes as a model pathogen. A total of eight L. monocytogenes strains, including WT strains, virulence gene-disrupted mutant strains (lap mutant and ⌬inlA and ⌬secA2 strains), and the respective complemented strains, were tested (Table 1). A total of 1,200 scatter patterns (50 scatter patterns/strain) were captured in three independent experiments for the WT, mutant, and complemented strains on BHIA and LBA. The scatter patterns of all of the mutant strains, i.e., the lap mutant, ⌬inlA, and ⌬secA2 strains, were profoundly different from that of the WT (Fig. 1a). The calculated positive predictive values (PPVs) for the strains varied from 93.7% ⫾ 6.2% to 97.3% ⫾ 2.1% when the colonies were grown on BHIA and from 99.1% ⫾ 0.1% to 100% ⫾ 0% when they were grown on LBA (Table 2). The strength of agreement (kappa value) for classification of the scatter images for each group (WT and mutants) was very good (mean kappa ⫾ standard error [SE] ⫽ 0.933 ⫾ 0.015), and the 95% confidence interval was 0.905 to 0.962. Similarly, the negative predictive value (NPV), specificity, and sensitivity were also very high (⬎91%), indicating that a substantial difference exists between the scatter patterns of WT and mutant strains. The scatter patterns of all mutant strains exhibited the prominent feature of a circular ring around the center, whereas the WT strains exhibited spokes and complex scatter patterns on BHIA (Fig. 1a). However, on LBA, very few scatter features (Zernike moment, rings and spokes; and Haralick texture, granularity) were visible compared to the scatter patterns that originated on BHIA. Intriguingly, most of the scatter features of complemented strains (i.e., lap mutant lap⫹, ⌬inlA inlA⫹, and ⌬secA2 secA2⫹ strains) were restored and were found to be qualitatively similar to those of the WT, indicating that the presence or absence of the particular virulence-associated proteins examined in this study specifically affected the scatter patterns. We also did not observe any substantial differences in the scatter features of the WT and WT Emr strains (Fig. 1a), suggesting that the observed differences in the scatter patterns were due to mutations in the virulenceassociated genes. This also further ruled out the contribution of erythromycin (10 ␮g/ml) in generating differential scatter patterns for lap mutant (KB208) colonies. Phase-contrast microscopic measurement of single cells also revealed that the cell size of

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FIG 2 Effects of different brands of growth medium on L. monocytogenes F4244 (WT). (a) Scatter images of L. monocytogenes F4244 on different brands of brain heart infusion agar (BHIA). (b) Image analysis using a cross-validation (CV) matrix. A total of 60 images/medium brand were analyzed to calculate the CV matrix.

the secA2 mutant was 2.3-fold greater (P ⬍ 0.0001) than that of the WT, whereas the average cell sizes of the other mutants and the complemented strains were close to that of the WT (Fig. 1b). The scatter patterns generated in this part of the study also suggested that high temperatures (42°C) and antibiotics (erythromycin at 10 ␮g/ml) did not affect the scatter patterns of the WT and WT Emr strains (Fig. 1c), respectively. Observations of the effects of growth medium formulations from different vendors on scatter patterns are essential for generating reproducible scatter patterns. Thus, we tested the effects of different brands of BHIA, from BD, Acumedia, and Teknova, on the WT colony scatter patterns, and we found that the patterns overlapped considerably, resulting in a low PPV (71%) and suggesting that different brands of BHIA had negligible effects on colony scatter patterns (Fig. 2a and b). We also used secA2 mutants of L. monocytogenes DP-L4342 (serotype 1/2a) and Listeria innocua F4248 (Fig. 3) to observe if secA2 mutants of other Listeria serotypes and strains, apart from L. monocytogenes F4244 (serotype 4b), affect the scatter patterns of mutant colonies in comparison to their WT parent strains. For the ⌬secA2 strain (DP-L4342) derived from the parent strain L. monocytogenes 10403S (serotype 1/2a), the colony appearance was rough, but this characteristic was not observed in F4244 (serotype 4b) in the present study (Fig. 3). Similarly, the ⌬secA2 mutant of L. innocua F4248 did not exhibit a rough-colony phenotype but gen-




branes by use of either Pierce ECL Western blotting substrate (Thermo Scientific) on X-ray film or LumiGlo substrate (Cell Signaling, Danvers, MA) to measure chemiluminescence with a charge-coupled device (CCD) camera-enabled ChemiDoc XRS system (Bio-Rad). Statistical analyses. Statistical analyses were performed using oneway analysis of variance (ANOVA) with Tukey’s multiple comparisons (GraphPad Prism, version 6.1) to measure significant differences (P values of ⬍0.05) with high individual scores for cell size measurements, ELISA, and the coinfection study. Scatter image analysis of the WT, mutant, and complemented strains was performed by use of the image classifier algorithm as described in the image analysis section. The image analysis results are presented as mean percent scores ⫾ standard errors of the means (SEM) (Table 2) and were calculated by analyzing scatter patterns acquired from two independent experiments with two replicates (n ⫽ 4). The general agreement (kappa value) between the PPVs (classification accuracies) for the scatter images of WT and mutant colonies of L. monocytogenes was also calculated by using the online QuickCalcs open access program (GraphPad software).


erated a markedly different scatter pattern from that of the parental WT strain (Fig. 3). These findings suggest that the differential scatter patterns of the mutant strains are due to the lack of production of specific proteins resulting from gene disruption. Biophysical properties of WT and mutant colonies. To understand the biophysical properties of the WT and mutant colonies of L. monocytogenes, the ICMA (37) was used to provide simultaneous and correlated information about each bacterial colony by two different measurement modalities, i.e., colony elevation (height) and light transmission (absorbance). The colony heights of the WT and WT Emr strains were 113.4 ␮m and 102.3 ␮m, respectively, on BHIA, whereas the heights of the mutant strains (lap mutant, ⌬inlA, and ⌬secA2 strains) were within the range of 83.9 to 109.3 ␮m on BHIA (Fig. 4). Similarly, the heights of the colonies of the WT and WT Emr strains on LBA were 78.7 ␮m and 68.7 ␮m, respectively, and the heights of the mutant strains were within the range of 66.3 to 72.5 ␮m (Fig. 4). The ODs of the WT and mutant colonies were measured using an in-house-built photodiode circuit equipped with a 10⫻ light microscope objective. The optical density was measured as a fraction of the incident light absorbed by the colony and is inversely proportional to the transmittance. The optical densities of the WT and WT Emr strains on BHIA and LBA were in the ranges of 0.41 to 0.51 and 0.45 to 0.48, respectively (Fig. 4), whereas the optical densities of the mutant strains were in the ranges of 0.46 to 0.57 and 0.35 to 0.43, respectively (Fig. 4). The absorbance and height were lower at the edges than at the centers of the colonies for all tested strains. Such differences in the optical densities of colonies could be attributed to the growth of newly dividing cells at the


FIG 4 Biophysical characteristics (i.e., height, diameter, and optical density) of L. monocytogenes colonies grown on brain heart infusion agar (BHIA) and Luria-Bertani agar (LBA). The colony profiles and optical densities were measured with an integrated colony morphology analyzer (ICMA). The profiles of a minimum of 15 colonies for each strain were measured to interpret the colony profiles, and the line graphs represent averages for 15 colony measurements.

edges of colonies, where they have access to more nutrients and oxygen (41–43). Conclusively, the ICMA revealed differences of 20 to 30 ␮m in the colony heights of the cultures grown on BHIA compared to those grown on LBA, but there were no differences in the optical density. Moreover, we did not observe any substantial differences in the heights or ODs between the WT and mutant strains on either medium, suggesting that these biophysical parameters possibly do not contribute to the changes in the scatter pattern. Identification of WT and mutant strains during coculture experiments. The WT and mutant strains used in the coculture and other experiments were first tested for the production of virulence-associated proteins and were validated by Western blot analysis (Fig. 5a). All tested strains except KB208 (lap mutant) produced the 104-kDa LAP. Similarly, all strains except AKB301 (⌬inlA) produced the 80-kDa InlA protein. Since we do not have a specific antibody for the SecA2 protein, we tested the ⌬secA2 mutant by probing with the anti-NamA MAb C11E9 (44). Previous studies have confirmed that NamA (also known as MurA; 66 kDa) is one of the proteins needed for cell separation (45), and its


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FIG 3 Phase-contrast, colony, and scatter images of WT and mutant strains of L. monocytogenes serotype 4b (F4244) and serotype 1/2a (10403S) and of WT and mutant strains of L. innocua F4248 on brain heart infusion agar (BHIA). Colonies were allowed to grow for ⬎25 h to observe roughness in colony edges, whereas scatter patterns were captured when the colony size was close to 1 mm in diameter, at 24 h of incubation. Phase-contrast image magnification, ⫻1000; colony image magnification, ⫻100. See Table 1 for details on the strains.

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expression in the WT and mutants before the mixed-culture experiment. (b) Scatter patterns and colony profiles at two different incubation times to differentiate mixed cultures of L. monocytogenes WT Emr and KB208 (lap mutant) on BHIA supplemented with 10 ␮g/ml of erythromycin (Em). (c) Scatter patterns of mixed cultures of the L. monocytogenes WT Emr and KB208 (lap mutant) strains on BHIA. BARDOT-identified representative single colonies (C1, C3, C5, C18, C26, and C58) were validated by an ELISA (bar graph) using the anti-LAP MAb Em10. (d) Scatter patterns of mixed cultures of the WT and ⌬inlA strains on BHIA. BARDOT-identified representative single colonies (C1, C4, C32, C49, C57, and C61) were also validated by an ELISA (bar graph) using the anti-InlA MAb 2D12. Petri dish pictures were captured with the BARDOT system; small white dots show mixed colonies, and yellow dots depict colony numbers. Control, carbonate coating buffer; LAP, Listeria adhesion protein; InlA, internalin A; NamA, N-acetylmuramidase.

secretion to the cell surface is SecA2 dependent (46, 47). The WT Emr and lap mutant scatter patterns were profoundly different at 25 h of incubation compared to those at 21 h of incubation (Fig. 5b). To test the differentiation capabilities of BARDOT for cocultures, two sets of mixtures were prepared: one contained the WT Emr strain and the lap mutant, and the other contained the WT and ⌬inlA strains. The cultures were plated on BHIA, and the scatter patterns were captured and matched against the WT, WT Emr, lap mutant, and ⌬inlA scatter image libraries (Fig. 5c and d). Five to seven BARDOT-identified colonies were confirmed by using LAP-specific antibody-based ELISA, showing 100% accuracy (Fig. 5c). Similarly, the WT and ⌬inlA scatter patterns were profoundly different after 22 h of incubation on BHIA, and InlA-specific ELISA confirmed the BARDOT-identified colonies with 100% accuracy (Fig. 5d). In another experiment, the cross-validation matrix obtained after the analysis of the scatter images of the WT and mutant strains on tryptic soy agar containing yeast extract (TSAYE) also revealed a substantial difference in the scatter patterns (ⱖ90% PPV), even though the colonies were not grown on agar medium in the presence of antibiotics (data not shown). Identification of WT and mutant strains during coinfection of Caco-2 cells. To demonstrate the application of BARDOT in bacterial pathogenesis studies for differentiation and enumeration of WT and mutant colonies based on scatter patterns, an in vitro coinfection study was performed using the Caco-2 cell

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line, and a total of 844 scatter images were generated. The WT and mutant (lap mutant and ⌬inlA) colonies each generated profoundly different scatter patterns on BHIA (Fig. 6a). In the coinfection study with the WT and lap mutant strains, BARDOT revealed that the percentage of WT colonies (79.5%) was significantly (P ⫽ 0.0036) higher than that of lap mutant colonies (20.5%). Likewise, in the study with the WT and ⌬inlA strains, the percentage of WT colonies (92.5%) was significantly (P ⬍ 0.0001) higher than that of ⌬inlA colonies (7.5%) (Fig. 6b). These data clearly indicate that the BARDOT method unequivocally quantifies the ratio of WT and mutant strains on the same plates during a coinfection study, and they further demonstrate specific roles of LAP and InlA in adhesion to/invasion of Caco-2 cells, as the respective mutant strains showed reduced infection. As a control, we also assessed the adhesion capacity of individual strains in the monoinfection study, and the plate count data showed adhesion of the WT (1.3 ⫻ 105 CFU/ml) to be significantly (P ⫽ 0.02) higher than that of the lap mutant (2.5 ⫻ 104 CFU/ml) or the ⌬inlA strain (2.5 ⫻ 103 CFU/ml) (Fig. 6c) (24). Using an erythromycin-sensitive WT strain and the erythromycin-resistant lap mutant strain during the coinfection study, we also determined that 21.6% of the total adhered colonies on erythromycin-supplemented BHIA were the lap mutant strain (data not shown), which is very close to the 20.5% of lap mutant colonies identified with the BARDOT method (Fig. 6b).




FIG 5 Scatter pattern-based differentiation of L. monocytogenes F4244 (WT) and mutant colonies in mixed culture. (a) Western blot analysis to validate protein


LAP expression affects the scatter patterns of L. monocytogenes. (i) Effects of lap mutation on scatter patterns. The scatter patterns and phase-contrast images of colonies of WT and lap mutant strains on BHIA and LBA were examined to observe the effects of the LAP protein on the colony scatter patterns. The scatter patterns of the lap mutant strain were substantially different from those of the WT on both BHIA and LBA (Fig. 7a), although the WT and lap mutant colony heights and transmittance levels were negligibly different on BHIA. The colony heights of both strains were ⬃90 ␮m on BHIA but ⬃70 ␮m on LBA (Fig. 7a). The scatter patterns of both WT and lap mutant strains on BHIA were different from the scatter patterns on LBA. This finding is in congruence with a previous study in which changes in the LB medium composition (agar and yeast extract concentrations) were also found to affect the morphologies and scatter patterns of E. coli colonies (48). Principal component analysis (PCA) demonstrated distinct clustering of the WT and lap mutant strains, which reiterated the differences in the scatter patterns (Fig. 7b). Image analysis of scatter patterns revealed that PPVs for the WT and lap mutant strains were both 100% on BHIA and 99.8% and 98.9%, respectively, on LBA (data not shown). The high PPVs indicate substantial differences in the scatter patterns of the WT and lap mutant strains. Western blot analysis with the cell wall (CW) and intracellular (IC) protein fractions confirmed the presence of LAP in the WT and its absence in the lap mutant strain (data not shown). Thus, it can be concluded that the absence of LAP was responsible for the generation of the distinguishing scatter features between the WT and the mutant KB208 (lap mutant) strains. (ii) Variable lap gene expression. Previously, we demonstrated that strains expressing various levels of LAP exhibited variable adhesion and virulence properties in a cell culture model


(49). Here we examined the colony scatter patterns of three L. monocytogenes strains, i.e., H4, F4244, and F4262, with differential LAP expression. The scatter patterns and colony profile measurements for the three strains (Fig. 8a) were captured after growth on BHIA and LBA. On LBA, F4244 and F4262 exhibited similar scatter patterns that were substantially different from that of H4. However, on BHIA, there was no profound difference in the scatter patterns or colony profiles between the strains (Fig. 8a). Image analysis using a cross-validation matrix confirmed the higher classification rates of the scatter patterns on LBA (96.4% to 100%) than on BHIA (88.2% to 93.4%) for all three strains. The PPVs for the scatter images of F4244, F4262, and H4 on BHIA were 93.4, 88.2, and 84.4%, respectively, whereas the corresponding values were 99.8, 96.4, and 100% on LBA (Fig. 8b). L. monocytogenes H4 is a significantly (P ⬍ 0.0001) higher LAP producer than F4244 and F4262, as demonstrated by Western blotting (Fig. 8c) and ELISA (Fig. 8d). We were interested in correlating the differences in the scatter patterns of the 4b serotype strains with the amounts and types of synthesized cell wall-associated proteins. SDS-PAGE analysis with the normalized cell wall protein fractions indicated more intense bands for F4244 and H4 than for F4262 (Fig. 8c). Western blotting and ELISA confirmed the high LAP production of the H4 strain (Fig. 8d) (49). This variation in the amount of protein synthesis may be one of the factors that contributed to the differences observed in the scatter patterns of the serotype 4b strains. DISCUSSION

Endeavors to correlate the bacterial genotype with phenotype for a better understanding of bacterial etiology or complex problems of evolutionary, ecological, pathogenic, or biochemical significance have placed a burden on the methodologies needed for the screen-


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FIG 6 BARDOT-based identification of L. monocytogenes WT and mutant strains (mixed at a 1:1 ratio) during coinfection of Caco-2 cells. (a) Representative plate pictures showing scatter patterns of mixed colonies of WT and mutant strains (lap mutant and ⌬inlA strains) on BHIA. (b) Enumeration of adhered colonies of the WT and lap mutant strains or the WT and ⌬inlA strains after coinfection of Caco-2 cells from the dilutions that were countable. Data are presented as percentages of WT and mutant colonies per plate. (c) Enumeration of each bacterial strain (WT, ⌬inlA, and lap mutant strains) after adhesion to Caco-2 cells during monoinfection. Plate pictures represent the numbers of adhered colonies of WT and mutant strains plated at the same dilution after monoculture infection of Caco-2 cells. Three independent mono- and coinfection experiments were performed, with strains plated on BHIA in duplicate (n ⫽ 6) to enumerate and capture the scatter patterns of colonies. The Mann-Whitney t test was performed to find the significant differences (P ⬍ 0.05) in bacterial adhesion for panel b.

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ing and isolation of mutant bacteria. In the field of biotechnology, mutations draw great interest in the directed evolution of proteins exhibiting improved or altered function (50). In general, bacterial mutant screening procedures rely on the analysis of the phenotype of each mutant independently, either on plates with suitable enzymatic substrates, such as X-Gal (5-bromo-4-chloro-3-indolyl␤-D-galactopyranoside) assays for the detection of ␤-galactosidase activity, or by measuring the enzyme activity in cell lysates or using an antibiotic selection marker. The major limitation with these methods of mutant screening is that they are arduous, label dependent, and invasive, requiring cell lysis for nucleic acid preparation or protein extraction. In the present study, we used a noninvasive, label-free, laserbased mutant colony screening method. Our results demonstrate that virulence gene-associated mutant colonies of L. monocytogenes (lap mutant, ⌬inlA, and ⌬secA2 strains) revealed profound differences (⬎93% PPV) in the colony scatter pattern compared to the WT strains on BHIA or LBA. In the complemented mutant

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strains (lap mutant lap⫹, ⌬inlA inlA⫹, and ⌬secA2 secA2⫹ strains), the scatter features were restored to patterns similar to the scatter patterns of the WT. Previous studies suggested that a bacterial colony behaves like a self-engineered spatial organization (i.e., multicellular organism) that displays intricate communication capabilities and social intelligence (51, 52) to cope with and respond to the surrounding environment (53–55). During such cooperation, genotypic variations in the individual bacterium amplify the overall variation of the colony by a million- or billionfold, which affects the colony morphotype (53). Moreover, continued binary fission of single bacterial cells results in colony formation, first in two-dimensional space and subsequently in three-dimensional space (43), and variations at the single-cell level define the morphotype of a bacterial colony (56). 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




FIG 7 Effects of LAP expression on scatter patterns of L. monocytogenes strains F4244 (WT) and KB208 (lap mutant) on BHIA and LBA. (a) Scatter pattern-, colony dimension-, and integrated colony morphology (height and transmittance)-based analysis of the strains. Scatter images and colony dimensions are for two different colonies obtained from the same experiment. (b) Principal component analysis (PCA) based on the similarity matrices generated by the image classifier algorithm for the colony scatter images of the L. monocytogenes WT and KB208 (lap mutant) strains on BHIA (left) and LBA (right). Each dot represents a colony scatter image.


expect that any mutation in the genes involved in the biochemical pathways for substrate utilization will also result in differential scatter patterns. Mutation in SecA2 results in a rough-colony phenotype (29), and this effect may be serotype dependent. Therefore, we also studied the effect of secA2 mutation on the colony scatter phenotypes of L. monocytogenes (serotypes 4b and 1/2a) and L. innocua strains. SecA2 is part of the Sec translocase system and thus plays an important role in virulence attributes by translocating proteins from the site of synthesis to the cell surface in some Gram-positive bacteria (28, 29, 46, 57). We observed an about 2.3-fold increase (Fig. 1a and b) in the single-cell size of the L. monocytogenes secA2 mutant (AKB103) that resulted in a scatter pattern distinct from that of the WT strains on both BHIA and LBA plates. Our results support previous observations that mutations in SecA2 produce elongated cells due to the defective translocation of muramidase enzymes, which are required for cell septation and division, across the membrane (29, 45). These findings suggest that the differential scatter patterns of the mutant strains are due to the lack of production of specific proteins due to gene disruption. Furthermore, this study highlights that the light scattering sensor is sensitive to genetic alterations in bacteria that affect protein expression and are magnified by 100 to 1,000 millionfold in single colonies of 100 to 1,000 million cells. In our previous study (5), we also observed that Salmonella enterica serovars with distinct pulsed-field gel electrophoresis (PFGE) fingerprints generated distinguishing colony scatter patterns. Therefore, it can be delineated that genotype defines the colony phenotype (scatter phenogram), which opens the scope of the BARDOT method for rapid screening of mutant strains.


In another set of experiments, we tested the feasibility and applicability of BARDOT for differentiating mixed colonies of the WT with the lap mutant and ⌬inlA strains based on signature colony scatter patterns for cocultures, and also after coinfection of the Caco-2 cell line. LAP is an alcohol acetaldehyde dehydrogenase enzyme with moonlighting functions (31, 58) that can help bacteria to catalyze ethanol to ethanoic acid, and it also functions as an adhesin and epithelial paracellular translocation factor to initiate interactions with host epithelial cells. InlA is an established member of the internalin protein family that uses E-cadherin for adhesion to and invasion of host cells (59). In the coculture assay, both the lap mutant and ⌬inlA strains generated scatter patterns that were markedly different from that of the WT on BHIA medium. The identification efficiency of BARDOT (5) for the mixed culture was also validated with LAP- and InlA-specific antibodies by use of ELISA (Fig. 5). Coinfection experiments were also performed with the WT and mutant strains to enumerate the relative abundances of these strains during infection in vitro to determine the importance of a specific protein (biomolecule) in pathogenesis (60). In the Caco-2 coinfection study, BARDOT was able to identify and enumerate mixed colonies of the WT and mutant strains (lap mutant and ⌬inlA), which were visually indistinguishable on the BHIA plate. Such capabilities of the BARDOT method could be used to make scatter pattern-based informed decisions to distinguish/screen mutant colonies during routine bacterial pathogenesis/genetic manipulation studies. The ability of BARDOT to distinguish bacterial colonies was further strengthened when scatter patterns of the lap mutant strain were shown to be distinct from those of the WT on both BHIA and LBA, though the biophys-


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FIG 8 BARDOT-generated scatter patterns distinguish strains of L. monocytogenes serotype 4b. (a) Colony profiles and scatter patterns of the strains on BHIA and LBA. (b) Quantitative analysis of scatter patterns based on a cross-validation matrix, using the image classifier algorithm. (c) SDS-PAGE as a loading control and Western blot analysis to assess the levels of LAP production from three strains of L. monocytogenes serotype 4b. (d) ELISA revealed variable LAP expression in the L. monocytogenes strains. Control, carbonate coating buffer. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple-comparison test, and asterisks indicate significant differences (P ⬍ 0.0001) in the average scores for fluorescence from three independent experiments with three replicates (n ⫽ 9).

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ACKNOWLEDGMENTS We acknowledge the technical assistance of Zhenjing Tang, Wen Lv, and Luping Xu and thank Marcelo Mendonca for supplying the antibody to InlA. We have no conflicts of interest to disclose. This research was supported by a cooperative agreement with the Agricultural Research Service of the U.S. Department of Agriculture (project 1935-42000-072-02G) and the Center for Food Safety Engineering at Purdue University.

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FUNDING INFORMATION This work was funded by U.S. Department of Agriculture (USDA) (193542000-072-02G).

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ical properties (colony height, diameter, transmittance, and reflectance) remained unchanged. This finding is in congruence with a previous study in which changes in the LB medium composition (agar and yeast extract concentrations) were also found to affect the growth morphologies and scatter patterns of E. coli colonies (48). In our previous study, it was demonstrated that three L. monocytogenes strains (i.e., H4, F4244, and F4262) of serotype 4b express various levels of LAP and exhibit variable adhesion and pathogenesis in a cell culture model (49). In the present study, these strains also generated differentiating scatter patterns on both BHIA and LBA media. The high-LAP-producing strain of L. monocytogenes serotype 4b (H4) also exhibited scatter patterns profoundly different from those of the other two, low-LAP-producing strains (F4244 and F4262) on LBA. This could be explained because LBA is a minimal medium which is known to induce LAP production (61), resulting in even more LAP production by the high-LAP-producing strain (H4) and giving a markedly different scatter pattern. However, upregulation of other gene products in LBA for the H4 strain cannot be ruled out. Compared to those of the WT colonies, the colony heights and optical densities of the mutant strains were similar on both BHIA and LBA media. Taken together, it can be concluded that BARDOT can efficiently differentiate mutant strains from WT colonies of L. monocytogenes and may possibly be applied for the screening of other bacterial mutants. In summary, BARDOT-based optical scatter sensing revealed differential scatter patterns of WT and virulence gene-associated mutant (i.e., lap mutant, ⌬inlA, and ⌬secA2 strains) colonies of L. monocytogenes and successfully differentiated the mutants and WT strains in mixed cultures and during coinfection of Caco-2 cells. To the best of our knowledge, this is the first report of a light scattering sensor being used for differentiation of mutant and WT colonies of L. monocytogenes, and the loss of a specific virulence gene resulted in a differential colony scatter phenotype. The data presented in this study suggest that BARDOT can be used as a user-friendly, label-free, and noninvasive tool for rapid highthroughput screening of mutant strains of L. monocytogenes, and possibly other bacteria.



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