Journal: PLoS ONE

Article Title: Forced Resurgence and Targeting of Intracellular Uropathogenic Escherichia coli Reservoirs

doi: 10.1371/journal.pone.0093327

Figure Lengend Snippet: ( A ) Growth of UTI89 in modified M9 medium containing increasing concentrations of chitosan (0.00002, 0.0002, 0.002%) or phosphate buffer (pH 4.5). Data shown are representative of three independent experiments carried out in triplicate. ( B ) Graph shows chitosan effects on biofilm production by UTI89 relative to untreated controls, as measured by microtiter plate assays. Data represent mean results ± SEM from three separate experiments performed in quadruplicate. Inset shows an example of filamentous bacteria in 0.002% chitosan; scale bar, 10 μm. Chitosan (0.0002 or 0.002%) effects on ( C ) UTI89 growth in RPMI medium during a 2-h incubation with 5637 BECs, ( D ) bacterial attachment to BECs, ( E ) bacterial invasion of BECs, and ( F ) bacterial survival within BECs over a 14-h period were quantified relative to buffer-treated controls. The data in F were normalized by dividing the numbers of surviving intracellular bacteria recovered from BECs after the 14-h incubation with gentamicin by the numbers of invading bacteria present after the 2-h incubation with gentamicin. Graphs show mean results ± SEM from three independent experiments performed in triplicate. * P <0.05, ** P <0.01, *** P <0.001 versus buffer-treated controls, as determined by Student’s t test with Welch’s correction when appropriate.

Article Snippet: The 5637 bladder epithelial cell line (ATCC, HTB-9) was grown in complete RPMI medium (supplemented with 10% fetal bovine serum, Sigma-Aldrich) and host cell adherence, invasion and intracellular survival assays were carried out as previously described , , , .

Techniques: Modification, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Development of mass allelic exchange, a technique to enable sexual genetics in Escherichia coli

doi: 10.1073/pnas.2105458119

Figure Lengend Snippet: A gain-of-function screen for in vitro pod formation. ( A ) Schematic of the screen. Orange shapes represent individual bladder epithelial cells, light blue represents nuclei, and green represents aggregates of fluorescent E. coli . In the actual screen, 5637 cells at ∼10 6 cells per well in a 6- or 12-well plate are infected with E. coli (either a transconjugant library or individual strains) at an MOI of 10 for 2 h, then a high-dose (100 μg/mL for 2 h) and a low-dose (10 μg/mL overnight) gentamycin treatment is used to kill extracellular bacteria; then, gentamycin is removed, 0.025% saponin is added for 30 min then washed away, and cells are observed for pod formation. A representative image of green fluorescence ( E. coli ) 6 h after saponin treatment and removal is shown at the bottom right of the panel. (Scale bar, 100 μm.) ( B ) Whole-genome sequencing of 5 pod-forming transconjugants (red) and 18 additional transconjugants (magenta and black; see text for details) ( y axis). The transferred regions for each clone are shown in red or magenta (pod-forming transconjugants) or black (non-pod-forming transconjugants), based on the MG1655 coordinates ( x axis). The left-most and right-most vertical dotted lines indicate the common region of UTI89 transferred to the clones depicted in red. The middle vertical dotted line delimits the left border of the common region of UTI89 transferred to all pod-forming transconjugants. ( C ) Genetic map of the common overlapping region for all pod-forming transconjugants. Notations are as in Fig. 1 C . Magenta line between the UTI89 and MG1655 coordinate axes at the bottom denotes the common overlapping region of all pod-forming clones. ( D ) Representative images of the pod-forming assay. Each strain is represented by three images; the smaller images on the top are the DAPI (blue) and wheat germ agglutinin (orange) channels ( Left ) and GFP (green) ( Right ). The larger image is a merge of all channels. White arrows indicate identified large pods. Scale bar shown at the bottom is 50 μm for larger images. Scale bar (50 μm) for smaller images is in white in the MG1655/pChu GFP image. ( E ) Box-and-whisker plot of area (in pixels) for all intracellular structures identified for UTI89 strains. Log (base 10) of the area is indicated on the x axis, strain genotype on the y axis. Center line for each plot represents the median, boxes indicate the first and third quartiles, and whiskers are set at 1.5× the interquartile range. Open circles represent outliers outside the range of the whiskers. A violin plot is superimposed on each box, depicting the distribution of all the data. The vertical dotted line indicates 10,000 pixels. ns, P > 0.05; * P < 0.01; ** P < 0.001 (two-tailed Wilcoxon rank-sum test).

Article Snippet: The 5637 bladder epithelial cells were obtained from ATCC (product HTB-9).

Techniques: In Vitro, Infection, Fluorescence, Sequencing, Clone Assay, Whisker Assay, Two Tailed Test