Antimicrobial susceptibility of <i>V. cholerae</i> RND mutants.

<p>Antimicrobial susceptibility was determined using antibiotic and detergent gradient agar plates.</p>1<p>The length of the mutant bacterial growth across the 90×90 mm gradient plate normalized to 100 mm. Data represents the average of three or more experiments with the standard deviation in parenthesis. Unpaired t-test was used to determine significance.</p>†<p>p<0.001 compared to N16961-Sm;</p><p>*p<0.05 compared to Δ<i>vexB</i>;</p><p>p<0.05 compared to Δ<i>vexBD</i>;</p><p>¡p<0.05 compared to Δ<i>vexBDHK</i>. NG: no bacterial growth.</p

Infant mouse colonization assays with the RND efflux mutants.

<p>Competition assays were performed using the infant mouse colonization assay as described in the Materials and Methods. Infant mice were challenged with a ∼10⁵ cfu inoculum containing a mixture of wild-type and the indicated mutant at a ratio of 1∶1 (A) or 1∶100 (B). The competitive index was calculated as the ratio of mutant to wild-type recovered from the small intestine, corrected for the ratio of mutant to wild-type that was present in the inoculum. Each symbol represents one mouse. *The ΔRND mutant was not recovered from mice necessitating the calculation of a theoretical CI as described in the Materials and Methods. Mean and standard deviation are indicated by horizontal bars. Significance was determined using the Mann-Whitney U t-test. (1) p<0.01.</p

Bacterial strains, Plasmids, and Oligonucleotides.

<p>Bacterial strains, Plasmids, and Oligonucleotides.</p

CT and TCP production by RND mutants.

<p>CT and TCP production in the indicated strains was determined following growth under AKI conditions. CT (A) and TcpA (B) were detected by CT GM₁-ELISA and TcpA Western immunoblotting, respectively. Error bars represent the standard deviation of the mean from three or more experiments. Statistical analysis was performed by one-way ANOVA. *p<0.05 compared to wild-type (WT); **p<0.05 compared to all tested strains.</p

Colonization of the infant mouse small intestine by <i>V. cholerae</i> RND efflux mutants.

<p>Infant mice were challenged with ∼6×10⁶ cfu (A) or ∼8×10⁷ cfu (B) of the indicated <i>V. cholerae</i> mutant. Bacterial loads in the small intestine were assessed after overnight incubation. Means and standard deviation are indicated by horizontal bars. The Mann Whitney U t-test was used to determine significance. (1) p<0.05 compared to all tested strains; (2) p<0.05 compared to Δ<i>vexBDK</i>; (3) p<0.05 compared to the ∼6×10⁶ cfu (A) Δ<i>vexBDH</i> challenge.</p

Correlation between whole animal <i>in vivo</i> imaging with viable bacterial counts 24 hours after challenge.

<p>BALB/c mice (5/group) were challenged with 1×10⁶ CFU of FTLVS bearing the pXB173-lux reporter plasmid via the intranasal route in a total bolus volume of either 10 µl, 20 µl, 50 µl, or 100 µl. <b>Panel A</b>: Dissemination of FTLVS was then monitored 24 hrs later using an IVIS Spectrum whole animal imaging system. Images were collected at the indicated time points post-infection and were normalized to reflect photons per second per cm∧2/sr. (<b>Panel B</b>): Lungs were collected after imaging was completed for bacterial burden determination via dilution plating. Statistical analyses were performed via one-way ANOVA using a Bonferroni multiple comparisons posttest. Statistically significant differences are indicated as follows: p<0.05 (*) and p<0.01 (**).</p

Kinetic <i>in vivo</i> localization of luminescent FTLVS following intranasal dosing in titrated volumes of inocula.

<p>BALB/c mice (3/group) were challenged via the intranasal route with 1×10⁶ CFU of FTLVS-lux suspended in a volume of 10 µl, 20 µl, 50 µl, or 100 µl of sterile PBS. <b>Panel A</b>: All mice were then subjected to whole animal imaging using an IVIS Spectrum Imaging system at the indicated time points. Scaling intensity of all images was normalized and data are reported as photons/sec/cm∧2/sr. <b>Panel B</b>: All mice were weighed daily as a measure of disease-state. Statistical analysis was performed via 2-way ANOVA with Bonferroni post-tests. Significant differences between the 10 µl instillation volume group and all other groups are indicated toward the top of the graph and are color-coded. Significant differences between the 100 µl instillation volume group and either the 20 µl or 50 µl dose volume groups are indicated toward the bottom of the graph and are color-coded. The calculated p values are indicated as follows: p<0.05 (*), p<0.01 (**), and p<0.001 (***).</p

Pulmonary delivery of FTLVS-lux was more efficient under inhaled vs. parenteral anesthesia.

<p>BALB/c mice (5/group) were anesthetized using either inhaled isoflurane or parenterally-administered ketamine/xylazine and then challenged with either 1×10⁵ CFU FTLVS-lux in a volume of 50 µl (<b>Panel A</b>) or 1×10⁶ CFU FTLVS-lux in a volume of 100 µl (<b>Panel B</b>). Dissemination of FTLVS was monitored 24 hrs later using an IVIS Spectrum whole animal imaging system. Lungs were collected after imaging was completed for bacterial burden determination via dilution plating. All IVIS images were normalized to reflect photons per second per cm∧2/sr. Statistical analyses were performed using the student t test.</p

Genetic Map of pXB173-lux.

<p>pXB173-lux constitutively expresses the <i>Photorhabdus luminescens</i> luciferace (<i>lux AB</i>) and luciferase substrate (<i>luxCDE</i>) from the <i>Francisella groE</i> promoter. This vector also encodes a selectable marker for kanamycin resistance (<i>aph3′</i>).</p