Article Figures & Data
Figures
- FIG. 1.
Magnetic beads coated with GFP-tagged CBD proteins bind and immobilize bacterial cells. Ni-NTA agarose beads coated with CBD500 (A and B) immobilize L. monocytogenes Scott A cells onto their surface (C). Coating of M-270 Epoxy Dynabeads with CBD500 (D and E) and binding of Listeria cells to their surface (F) are shown. Bars in panels A to C are 20 μm, and those in panels D to F are 10 μm.
- FIG. 2.
Performance of CBD500-coated Ni-NTA magnetic agarose beads for separation of L. monocytogenes Scott A. Three variables were tested for their effects on recovery rates, i.e., (A) different bead concentrations (indicated as the absolute number used in the 200-μl assay volume (with 104 cells, 40-min incubation), (B) different incubation times (with 104 cells, 1.7 × 105 beads), and (C) different cell concentrations (with 1.7 × 105 beads, 40-min incubation).
- FIG. 3.
Influence of blocking agent and CBD type on binding and recovery with CBD-coated Dynabeads. (A) Immobilization of L. monocytogenes EGDe with CBD118-coated beads blocked with either BSA or Tris. (B) Separation of L. monocytogenes EGDe or Scott A after contact with beads coated with CBD118, CBD500, or a 1:1 mixture of both bead types or with uncoated, Tris-blocked control beads, respectively.
- FIG. 4.
CBD-coated beads do not lead to agglutination of cell-bead mixtures. Light microscopy images show the cross-linked agglomerates formed after incubation of anti-Listeria Dynabeads with cell suspensions of both L. monocytogenes Scott A (panel A) and EGDe (panel B), respectively. In contrast, beads coated with CBD500 (panel C) or CBD118 (panel D) did not show significant agglutination after incubation with Scott A and EGDe cells, respectively.
- FIG. 5.
Optimization of parameters for separation and recovery of Listeria cells with CBD-coated Dynabeads. CBD118 beads were tested with L. monocytogenes WSLC 1001 and EGDe, and CBD500 beads were used with L. monocytogenes Scott A and L. ivanovii WSLC 3009. Three variables were tested, i.e., different bead concentrations, indicated as the absolute number used in the 200-μl assay volume (with 104 cells, 40-min incubation time) (panels A and B), different incubation times (104 cells, 2 × 107 beads) (panels C and D), and different cell concentrations (2 × 107 beads, 40-min incubation) (panels E and F).
- FIG. 6.
CBD proteins from endolysins of B. cereus and C. perfringens bacteriophages for labeling and immobilization of host target bacteria. Panels: A, B. cereus cells fluorescently decorated by GFP-tagged CBD21; B, C. perfringens cells labeled by GFP-CBD3626 (see text for details); C, C. perfringens cells immobilized on CBD3626-coated Dynabeads; D, rates of immobilization and recovery of cells of the two organisms with Dynabeads coated with the respective CBD molecules.
Tables
- TABLE 1.
Efficiencies of recovery of different Listeria species and serovars from mixed bacterial suspensionsa
Strain (serovar) CBD type on beads Rate of recovery (mean ± SD)b L. monocytogenes EGDe (1/2a) CBD118 97.2 ± 0.9 WSLC 1001 (1/2c) CBD118 94.9 ± 1.8 Scott A (4b) CBD500 96.2 ± 0.8 WSLC 1042 (4b) CBD500 96.6 ± 1.4 WSLC 1363 (4b) CBD500 93.8 ± 0.9 L. ivanovii WSLC 3009 (5) CBD500 92.2 ± 1.8 L. innocua WSLC 2012 (6b) CBD500 98.6 ± 0.9 - TABLE 2.
Comparison of standard plating and magnetic separation with CBD118-coated beads for detection of L. monocytogenes EGDe in artificially contaminated food samples
Food sample and initial contamination (CFU/g) Result obtained with following conditions for selective enrichment culturea 6 h 24 h 48 h IDF/ISOb CBD-MS IDF/ISO CBD-MS IDF/ISO CBD-MS Sliced iceberg lettuce 0 − − − − − − 0.1 − − +++ +++ +++ +++ 1 (+) − +++ +++ +++ +++ 10 − + +++ +++ +++ +++ 100 − ++ +++ +++ +++ +++ Camembert soft cheese 0 − − − − − − 0.1 − − − − − + 1 − − ++ +++ ++ ++ 10 − − +++ +++ +++ +++ 100 + ++ +++ +++ +++ +++ Red smear cheese 0 − − − − − − 0.1 − − − + − + 1 − (+) − ++ − + 10 − − +++ +++ +++ +++ 100 − + +++ +++ +++ +++ Smoked salmon 0 − − − − − − 0.1 − − + ++ ++ ++ 1 − − ++ ++ ++ +++ 10 − + +++ +++ +++ +++ 100 + ++ +++ +++ +++ +++ Minced meat 0 − − − − − − 0.1 − + + + +++ +++ 1 − + + +++ +++ +++ 10 − + +++ +++ +++ +++ 100 − ++ +++ +++ +++ +++ Turkey breast 0 − − − − − − 0.1 − + + ++ − + 1 − − +++ +++ ++ +++ 10 − + +++ +++ +++ +++ 100 + + +++ +++ +++ +++ Milk (pasteurized) 0 − − − − − − 0.1 − − +++ +++ +++ +++ 1 − − +++ +++ +++ +++ 10 − + +++ +++ +++ +++ 100 + ++ +++ +++ +++ +++ - TABLE 3.
Comparison of standard plating and magnetic separation with CBD500-coated beads for detection of L. monocytogenes Scott A in artificially contaminated food samples
Food sample and initial contamination (CFU/g) Result obtained with following conditions for selective enrichment culturea 6 h 24 h 48 h IDF/ISOb CBD-MS IDF/ISO CBD-MS IDF/ISO CBD-MS Sliced iceberg lettuce 0 − − − − − − 0.1 − − ++ +++ +++ +++ 1 − − +++ +++ +++ +++ 10 − + +++ +++ +++ +++ 100 − + +++ +++ +++ +++ Camembert soft cheese 0 − − − − − − 0.1 − − + +++ + + 1 − + +++ +++ + − 10 + + +++ +++ ++ ++ 100 + ++ +++ +++ +++ +++ Red smear cheese 0 − − − − − − 0.1 − − + +++ + + 1 − − +++ +++ +++ +++ 10 − + +++ +++ +++ +++ 100 − + +++ +++ +++ +++ Smoked salmon 0 − − − − − − 0.1 − − − + + ++ 1 − − + + ++ +++ 10 − + +++ +++ +++ +++ 100 + ++ +++ +++ +++ +++ Minced meat 0 − − − − − − 0.1 − − − − − − 1 − − − + ++ +++ 10 − − − ++ +++ +++ 100 + ++ ++ +++ +++ +++ Turkey breast 0 − − − − − − 0.1 − − − + + ++ 1 − − − + + ++ 10 − − + ++ ++ +++ 100 − + ++ +++ +++ +++ Milk (pasteurized) 0 − − − − − − 0.1 − − − + − + 1 − − + + − + 10 + + + +++ + ++ 100 + +++ +++ +++ +++ +++ - TABLE 4.
Validation of CBD-MS plating with naturally contaminated food samples
Food type or parameter No. of samples positive/no. tested (% positive)a CBD-MS plating Standard plating method Meat and meat products 6/26 (23) 5/26 (19) Poultry 9/13 (69) 9/13 (69) Fish and seafood 25/92 (27) 23/92 (25) Dairy products 4/110 (4) 4/110 (4) Delicatessen salads 1/34 (3) 1/34 (3) Total positive (Listeria spp.) 45/275 (16) 42/275 (15) Positive for L. monocytogenes 28/275 (10) 26/275 (9) Relative performance (%)b 100 93 Total time required (h) 48c 96d ↵ a Values were rounded to the next whole number.
↵ b Relative performance was set at 100% for the system which detected the most positive samples.
↵ c Selective enrichment for 22 h, CBD-MS, and plate incubation for 24 h.
↵ d International IDF standard/ISO norm (selective enrichment for 48 h and plate incubation for 48 h).
