Journal: PLoS ONE

Article Title: Variable Responses to Carbon Utilization between Planktonic and Biofilm Cells of a Human Carrier Strain of Salmonella enterica Serovar Typhi

doi: 10.1371/journal.pone.0126207

Figure Lengend Snippet: A total of 190 carbon substrates were tested. A: Alcohol, B: Amide, C: Amine, D: Amino acid, E: Carbohydrate, F: Carboxylic acid, G: Ester, H: Fatty acid, I: Polymer. Y-axis indicates the percentage of carbon utilized in planktonic, biofilm and biofilm inducing S . Typhi bacterial growth stages. X-axis shows the carbon category for each carbon substrate tested. The Venn diagram was obtained based on the Average Growth Curve (AUC) area and was classified into a combination of 6 different bacterial growth stages: growth only in planktonic; only in biofilm; inducing biofilm formation; planktonic and biofilm only; planktonic and inducing biofilm formation only; biofilm and inducing biofilm formation only; all 3 stages of bacterial growth planktonic, biofilm and inducing biofilm formation. S . Typhi biofilm growth stage was tested using the 96-well peg lid on Phenotype MicroArray plate for 48 h. The biofilm inducing experiment was conducted using 0.5% crystal violet stain and absorbance was measured at wavelength OD 590nm every 6 h.

Article Snippet: In this study, differential carbon catabolism of the strain in planktonic and biofilm stages was measured using the high-throughput Biolog Phenotype MicroArray (PM) and Minimum Biofilm Eradication Concentration (MBEC) biofilm inoculator (96-well peg lid) [ ].

Techniques: Polymer, Microarray, Staining

Journal: PLoS ONE

Article Title: Variable Responses to Carbon Utilization between Planktonic and Biofilm Cells of a Human Carrier Strain of Salmonella enterica Serovar Typhi

doi: 10.1371/journal.pone.0126207

Figure Lengend Snippet: Fig 2A. Carboxylic acid; Fig 2B. Carbohydrate; Fig 2C. Amino acid; Fig 2D. Ester, fatty acid and polymer. Area under the growth curve values of substrates utilized by S . Typhi strain CR0044 was determined using Biolog Phenotype MicroArray plates PM1 and PM2. The maximal kinetic curve height was expressed as a grayscale ranging from 0 (light gray) to 44 (black) area under the curve units. Color highlights show differences between biofilm and planktonic S . Typhi; green (planktonic only), yellow (biofilm only), red (both planktonic and biofilm), purple (induced biofilm). Phenotypes < 0 were considered negative.

Article Snippet: In this study, differential carbon catabolism of the strain in planktonic and biofilm stages was measured using the high-throughput Biolog Phenotype MicroArray (PM) and Minimum Biofilm Eradication Concentration (MBEC) biofilm inoculator (96-well peg lid) [ ].

Techniques: Polymer, Microarray

Journal: Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology

Article Title: Povidone-Iodine Fails to Eradicate Chronic Suppurative Otitis Media and Demonstrates Ototoxic Risk in Mice

doi: 10.1097/MAO.0000000000003726

Figure Lengend Snippet: Minimum biofilm eradication concentration of chronic suppurative otitis media clinical isolates against povidone-iodine (n = 3 for each strain)

Article Snippet: Determination of Minimum Biofilm Eradication Concentration Overnight cultures of PA01, SA 25923, PA clinical isolates, and SA clinical isolates were diluted 1:1,000 in fresh LB medium and 150 μl of the dilution per well were added in a Minimum Biofilm Eradication Concentration (MBEC) Assay Biofilm Inoculator (Innovotech, Edmonton, Canada) with 96 wells.

Techniques: Concentration Assay

Journal: iScience

Article Title: Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa

doi: 10.1016/j.isci.2023.106843

Figure Lengend Snippet: An experimental setup to examine the influence of periodic disturbance on the expression of genes in the central quorum sensing network of P. aeruginosa (A) The central quorum sensing network in P. aeruginosa . Each section of the network is color coded: las regulon = yellow; rhl regulon = red; pqs regulon = blue; iqs regulon = gray. Each regulon produces its own autoinducer (different shapes outside of the cell) that binds to a receptor on the inside of the cell (denoted with dotted lines). Virulence factors measured in this study and regulated by one or more receptors are shown. Normal arrow indicates activation; blunt end arrow indicates repression. LasB (orange) is regulated by both the las and rhl regulons. HcnA (purple) is regulated by both the rhl and pqs regulons. (B) Experimental approach to growing and disturbing the structure of biofilms. P. aeruginosa biofilms were grown in an MBEC Inoculator device. The plate lid contains 96 rounded polystyrene pegs that allow for the attachment and formation of biofilms when immersed in liquid medium; the liquid medium contains a population of bacteria in the planktonic state. Biofilms were disturbed using linear shaking of a microplate reader at defined intervals (called shaking frequency) and amplitudes for 24 h. (C) Optimizing experimental conditions for growing biofilms. Biofilms were grown for 24 h whereupon the medium surrounding the peg was replaced with fresh medium (wash) or was not replaced (no wash). Biofilm density was quantified using a crystal violet assay (see ) and measured at OD 555 . Density of bacteria in the planktonic state was quantified using OD 600 . ∗ significant difference in OD measurement. No wash treatment: OD 600 - 24 h vs. 48 h (p < 0.001, two-tailed t-test); 48 h vs. 72 h (p = 0.026, two-tailed t-test). OD 555 - 24 h vs. 48 h (p = 0.024, two-tailed t-test); 48 h vs. 72 h (p = 0.002, two-tailed t-test). Wash treatment: OD 600 - 24 h vs. 48 h (p = 0.089, two-tailed t-test); 48 h vs. 72 h (p = 0.004, two-tailed t-test). OD 555 - 24 h vs. 48 h (p = 0.022, two-tailed t-test); 48 h vs. 72 h (p = 0.44, two-tailed t-test). Average and standard deviation from ≥4 biological replicates. (D) Schematic of our experimental approach to disturb the spatial structure of biofilms and measure the impact on the expression of genes regulated by quorum sensing. “Wash” encompasses placing the biofilms affixed to the plate lid in fresh medium for 10 s to remove unadhered bacteria followed by placing the biofilms in additional fresh medium. Thus, disturbance occurs in fresh King’s A medium.

Article Snippet: Biofilms were grown in MBEC biofilm inoculator plates (Innovotech, Edmonton, AB, Canada).

Techniques: Expressing, Activation Assay, Crystal Violet Assay, Two Tailed Test, Standard Deviation

Journal: iScience

Article Title: Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa

doi: 10.1016/j.isci.2023.106843

Figure Lengend Snippet: An intermediate disturbance frequency can reduce the expression of virulence factors in the las , rhl , and pqs regulons (A) Density of bacteria in the biofilm and planktonic states after 24 h of disturbance at the indicated frequency (0.3 mm amplitude). A significant (∗) reduction in biofilm density was observed at 12/h (p = 0.0114, Mann-Whitney (Shapiro-Wilk; p = 0.0022)), as compared to the undisturbed (0/h) control. A significant difference in the density of bacteria in the planktonic state was observed at all disturbance frequencies measured (p ≤ 0.0059, Mann-Whitney (Shapiro-Wilk; p < 0.0001) as compared to 0/h). Average and standard deviation from ≥6 biological replicates. (B) Effect of periodic disturbance on the expression of genes in the las regulon. There was a significant (∗) reduction in the expression of lasR , lasA , lasB , and aprA at a disturbance frequency of 12/h as compared to 0/h (p < 0.026, two-tailed t-test, all p values in Table S2 ). In panels B–D, average and standard deviation from ≥4 biological replicates. Δ C t data in Figure S2 . (C) Effect of periodic disturbance on the expression of genes in the rhl regulon. There was a significant (∗) reduction in the expression of rhlR , rhlA , lasB , and hcnA at 12/h as compared to 0/h (p < 0.041, two-tailed t-test, all p values in Table S2 ). A significant increase in the expression of rhlA was observed at 3/h (p = 0.0039, two-tailed t-test). (D) Effect of periodic disturbance on the expression of genes in the pqs regulon. There was a significant (∗) reduction in the expression of pqsR and hcnA (p < 0.022, two-tailed t-test, all p values in Table S2 ), but not phzM (p = 0.107), as compared to 0/h.

Article Snippet: Biofilms were grown in MBEC biofilm inoculator plates (Innovotech, Edmonton, AB, Canada).

Techniques: Expressing, MANN-WHITNEY, Standard Deviation, Two Tailed Test

Journal: iScience

Article Title: Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa

doi: 10.1016/j.isci.2023.106843

Figure Lengend Snippet: Periodic disturbance can result in a reduction in the expression of virulence factors across multiple disturbance amplitudes (A) The effect of different disturbance amplitudes on the density of bacteria in the biofilm and planktonic state when disturbed at a frequency of 6/h. A significant (∗) increase in the density of bacteria in the planktonic state was observed at an amplitude of 0.3 mm (p < 0.0001, Mann-Whitney (Shapiro-Wilk; p = 0.0166) compared to 0/h). For panels A–C, biofilm density was measured using a crystal violet assay and OD 555 ; density of bacteria in the planktonic state was measured using OD 600 . For panels A–C, average and standard deviation from ≥4 biological replicates. (B) The effect of disturbance amplitude on the density of bacteria in the biofilm and planktonic state when disturbed at a frequency of 12/h. A significant (∗) reduction in biofilm density was observed at an amplitude of 0.3 mm (p = 0.0057, Mann-Whitney (Shapiro-Wilk; p = 0.0074) compared to 0/h). A significant increase in the density of bacteria in the planktonic state was observed at amplitudes of 0.1 mm and 0.3 mm (p < 0.0001, two-tailed t-test, compared to 0/h). Δ C t data in Figure S5 . (C) The effect of disturbance amplitude on the density of bacteria in the biofilm and planktonic state when disturbed at a frequency of 15/h. A significant (∗) reduction in biofilm density was observed at an amplitude of 0.3 mm (p = 0.0095, Mann-Whitney (Shapiro-Wilk; p = 0.0125) compared to 0/h). A significant increase in the density of bacteria in the planktonic state was observed at amplitudes of 0. and 0.3 mm (p < 0.03, two-tailed t-test, compared to 0/h). (D) The effect of disturbance amplitude on the expression of select virulence factors from the las , rhl , and pqs regulons when biofilms were disturbed at 6/h. Expression of aprA (p < 0.035, two-tailed t-test, compared to 0/h) and rhlA (p < 0.044, data points behind phzM at 0.1 and 0.2 mm) were significantly (∗) reduced at amplitudes of 0.1 and 0.2 mm. In panels D–F, average and standard deviation from ≥4 biological replicates; the inset shows the average fold change in expression for all virulence factors measured Data point colors (bottom right) are used consistently for panels D–F. All p values in Table S2 ; Δ C t values in Figure S5 . (E) The effect of disturbance amplitude on the expression of select virulence factors from the las , rhl , and pqs regulons when biofilms were disturbed at a frequency of 12/h. Expression of aprA (p < 0.01, Mann-Whitney (Shapiro-Wilk; p = 0.0086), compared to 0/h) and rhlA (p < 0.041, two-tailed t-test) were significantly (∗) reduced at all amplitudes tested. Expression of phzM was significantly reduced at an amplitude of 0.2 mm (p = 0.014, Mann-Whitney (Shapiro-Wilk; p = 0.0009)). (F) Left: The effect of disturbance amplitude on the expression of select virulence factors from the las , rhl , and pqs regulons when biofilms were disturbed at a frequency of 15/h. Expression of aprA (p < 0.002, two-tailed t-test, compared to 0/h) , rhlA (p < 0.007, Mann-Whitney (Shapiro-Wilk; p = 0.0305), and phzM (p < 0.012, two-tailed t-test) was significantly (∗) reduced at amplitudes of 0.1 and 0.2 mm. Right: The main quorum sensing network in P. aeruginosa . Each section of the network is color coded to correspond to the virulence factor gene being measured in panels D–F. las regulon = yellow; rhl regulon = red; pqs regulon = blue.

Article Snippet: Biofilms were grown in MBEC biofilm inoculator plates (Innovotech, Edmonton, AB, Canada).

Techniques: Expressing, MANN-WHITNEY, Crystal Violet Assay, Standard Deviation, Two Tailed Test

Journal: iScience

Article Title: Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa

doi: 10.1016/j.isci.2023.106843

Figure Lengend Snippet: Removal of lasR can augment the decrease in expression of select virulence factors at a disturbance frequency of 12/h (A) Effect of disturbance at 0/h and 12/h (0.3 mm amplitude) on Δ lasR , Δ rhlR , Δ pqsR , and Δ lasR /Δ rhlR knockout strains. ∗ indicates statistically different from the wild-type strain (WT). At 0/h, there was an increase in the density of both the biofilm and bacteria in planktonic state for the Δ pqsR strain (p ≤ 0.0012, Mann-Whitney (p ≤ 0.0038, Shapiro-Wilk), and a decrease in the density of the biofilm in the lasR /Δ rhlR strain (p = 0.0176, Mann- Whitney (p = 0.0003, Shapiro-Wilk). At 12/h, the density of the biofilm was greater than WT for the Δ lasR , Δ rhlR , and Δ lasR /Δ rhlR strains (p ≤ 0.0252 Mann-Whitney (p = 0.0231, Shapiro-Wilk). In addition, the density of bacteria in the planktonic state was greater than WT in the Δ rhlR and Δ lasR /Δ rhlR strains (p ≤ 0.0002, two-tailed t-test). Average and standard deviation from ≥4 biological replicates. (B) Effect of removing receptors in the quorum sensing regulons on gene expression of select receptors and effectors. In this panel, gene expression of WT at 0/h is compared to each knockout strain at 0/h ∗ indicates significant difference (p < 0.02, each p value in Table S5 ). For panels B–E, average and standard deviation from ≥4 biological replicates and Δ C t data in Figure S6 . (C) Effect of disturbance at 12/h on gene expression in knockout strains as compared to the WT strain in the 0/h condition. ∗ indicates significant difference (p < 0.04, each p value in Table S5 ). (D) Effect of disturbance at 12/h on gene expression in knockout strains as compared to each respective knockout strain at 0/h ∗ indicates significant difference (p ≤ 0.05, each p value in Table S6 ). (E) Effect of disturbance at 12/h on gene expression in knockout strains as compared to the WT strain at 12/h ∗ indicates significant difference (p < 0.04, each p value in Table S5 ).

Article Snippet: Biofilms were grown in MBEC biofilm inoculator plates (Innovotech, Edmonton, AB, Canada).

Techniques: Expressing, Knock-Out, MANN-WHITNEY, Two Tailed Test, Standard Deviation

Journal: Journal of Clinical Microbiology

Article Title: In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

doi: 10.1128/JCM.02861-15

Figure Lengend Snippet: Effect of nonantibiotic agents on a preformed biofilm produced by representative laboratory isolates of bacteria a

Article Snippet: After final dilutions, 150 μl was transferred to a 96-well minimum biofilm eradication concentration (MBEC) microtiter plate (Innovotech, Inc., Edmonton, Alberta, Canada).

Techniques: Produced, Bacteria, Control

Journal: Journal of Clinical Microbiology

Article Title: In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

doi: 10.1128/JCM.02861-15

Figure Lengend Snippet: Effect of nonantibiotic agents on a preformed biofilm produced by bacteria isolated from mares with clinical disease

Article Snippet: After final dilutions, 150 μl was transferred to a 96-well minimum biofilm eradication concentration (MBEC) microtiter plate (Innovotech, Inc., Edmonton, Alberta, Canada).

Techniques: Produced, Bacteria, Isolation, Control

Journal: Journal of Clinical Microbiology

Article Title: In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

doi: 10.1128/JCM.02861-15

Figure Lengend Snippet: Efficacy of compounds tested as a percentage of total number of clinical isolates inhibited in multiple assays

Article Snippet: After final dilutions, 150 μl was transferred to a 96-well minimum biofilm eradication concentration (MBEC) microtiter plate (Innovotech, Inc., Edmonton, Alberta, Canada).

Techniques: Bacteria

Journal: Journal of Clinical Microbiology

Article Title: In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

doi: 10.1128/JCM.02861-15

Figure Lengend Snippet: Bioluminescent imaging of the equine uterus at 5 days postinoculation with lux-labeled P. aeruginosa. Luminescence was detected following repeated washing of the endometrial surface of the uterine body, indicating that the bioluminescent biofilm-like material produced by lux-labeled P. aeruginosa was strongly adherent and difficult to remove from the endometrium.

Article Snippet: After final dilutions, 150 μl was transferred to a 96-well minimum biofilm eradication concentration (MBEC) microtiter plate (Innovotech, Inc., Edmonton, Alberta, Canada).

Techniques: Imaging, Labeling, Produced