Journal: Communications Biology

Article Title: Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria

doi: 10.1038/s42003-021-01856-1

Figure Lengend Snippet: a Bioinformatic toolbox predicts PNAs that can target essential and non-essential genes in one or more Enterobacteriaceae including E. coli , K. pneumoniae (KPN), and S. enterica (STm). Here, PNAs targeting E. coli genome are identified and screened for internal off-targets. Candidates without off-targets are narrowed to those with homology among KPN and STm. Of the final 71 essential gene candidates and 243 non-essential gene candidates that met the thermodynamic requirements for experimental conditions, five and four PNAs targeting essential and non-essential gene respectively were randomly chosen for assessment. Most of the PNAs have homology to all three Enterobacteriaceae in this study, except α-folC, α-csgD, and α-fnr which are designed to be specific to E. coli , and α-recA which is specific to E. coli and STm. PNAs α-folC, α-ffh, α-lexA, α-acrA, α-recA, α-csgD, and α-fnr target novel pathways of metabolism, signal recognition, stress response, transport, stress response, biofilm formation, and metabolism respectively. PNAs α-gyrB and α-rpsD target novel genes in traditional antibiotic pathways. b Following PNA solid-phase synthesis, the product is purified using HPLC (representative chromatogram shown), verified by LCMS (representative spectra shown), and tested against a lab strain of E. coli (MG1655, growth curves for α-lexA and α-rpsD from Figure shown). c Antibiotic resistance characterization of clinical isolates of CRE E. coli , MDR E. coli , ESBL KPN, NDM-1 KPN, and MDR S . Typhimurium. (left) Antibiotic resistance characterization of clinical isolates used in this study. Letters “R”, “S”, and “I” indicate drug-resistance, sensitivity, and intermediate resistance respectively. Sensitive, intermediate, and resistant breakpoints are provided in Table . MIC90 antibiotic concentration ranges for clinical isolates are provided in Table . Nine antibiotics of varied mechanisms and classes were tested including penicillins (ampicillin, AMP), cephalosporins (ceftriaxone, FRX), carbapenems (meropenem, MER), aminoglycosides (gentamicin, GEN and kanamycin, KAN), tetracyclines (tetracycline, TET), fluoroquinolones (ciprofloxacin, CIP), quinolones (nalidixic acid, NXA), and phenicols (chloramphenicol, CHL). d Clinical isolate monotherapy testing is done by monitoring growth at OD 600nm over 16 h. Shown here are representative growth curves from Figure of four of the clinical isolates with 10 µM PNA: NDM-1 KPN and a PNA control, α-nonsense, MDR E. coli and α-ffh, CRE E. coli and α-rpsD, and MDR STm and α-acrA with at least three biological replicates and errors bars as standard deviation. e–f Normalized growth (ratio of optical density of treatment to no treatment at 16 h) of clinical isolates in the presence of treatment with 10 µM of the indicated PNA. PNAs targeting essential and non-essential genes and showing at least 50% growth inhibition are shown in panels e and f respectively with significance (represented by an asterisk, p value <0.05) determined relative to control nonsense PNA and no treatment respectively. All data shown are the average of at least three biological replicates with standard deviation shown as error bars. Grey circles indicate individual biological replicates. g PNA concentration ranges for 90% growth inhibition of each bacteria strain where shaded grey boxes indicate expected PNA homology for the given bacterial strain.

Article Snippet: E. coli MG1655 PNA growth experiments were carried out in M9 media (1x M9 minimal media salts solution (MP Biomedicals), 2.0 mM MgSO 4 , and 0.1 mM CaCl 2 in sterile water) with 0.4% glucose.

Techniques: Purification, Concentration Assay, Standard Deviation, Inhibition

Journal: Communications Biology

Article Title: Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria

doi: 10.1038/s42003-021-01856-1

Figure Lengend Snippet: a Using the STRING database of protein network interactions (data shown in Supplementary Figure ), we found a positive correlation, Pearson’s r = 0.908 and p < 0.001, between growth inhibition of PNA homologous with target bacteria and average node degree, the number of interactions of protein has in the average network. b – m Subsequent growth curves and bar plots show bacteria without treatment, or treatment with PNA alone (10 µM), or antibiotic alone, or PNA and antibiotic combined. Antibiotic concentrations were (from top to bottom) 2 µg/mL tetracycline (TET), 4 µg/mL gentamicin (GEN), and 8 µg/mL chloramphenicol (CHL). b, f, j The first column shows representative growth curves over 24 h of each treatment. Panels b–e and f–i show treatment of CRE E. coli with PNAs targeting essential and non-essential genes respectively. Panels j–m show treatment of ESBL KPN with PNAs targeting essential genes. All bar plots are the average OD (600 nm) of each treatment at 24 h normalized to no treatment at 24 h. S values above the bar plots were obtained using the Bliss Independence model and indicate synergistic interaction between PNA and antibiotic at 24 h, an asterix indicates significance at α = 0.05. Grey circles indicate individual biological replicates. Panels n–p show select heat maps of synergy values at 24 h of checkerboard combination assays (Figures - ). q Plotting the S values representing synergy against the target protein’s average node degree shows a negative correlation (Pearson’s r = −0.3017, p = 0.0314). All data shown are the average of at least three biological replicates with standard deviation shown as error bars.

Article Snippet: E. coli MG1655 PNA growth experiments were carried out in M9 media (1x M9 minimal media salts solution (MP Biomedicals), 2.0 mM MgSO 4 , and 0.1 mM CaCl 2 in sterile water) with 0.4% glucose.

Techniques: Inhibition, Standard Deviation

Journal: Communications Biology

Article Title: Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria

doi: 10.1038/s42003-021-01856-1

Figure Lengend Snippet: a Schematic showing the experimental setup for comparing PNA delivery into HeLa cells for treating an intracellular infection of Salmonella enterica serovar Typhimurium, strain SL1344 expressing GFP (STm-GFP), using either the bacterial T3SS inherent to STm-GFP (Bottom, T3SS-PNA) or CPP facilitated uptake (Top, Naked-PNA). All intracellular infection treatment experiments follow the general protocol of 45-min infection of HeLa cells 24 h after seeding, 75-min gentamicin treatment to remove extracellular bacteria, and 18-h incubation with treatment in media containing gentamicin to ensure an only intracellular infection model. For comparing T3SS delivered PNA to naked-PNA infection was done using STm-GFP at an MOI of 10. For the T3SS-PNA condition 10 µM of PNA was added during this 45-min infection stage; during this time the PNA is allowed to enter the Salmonella and subsequently be transported into the HeLa cell as it infects via its T3SS. For the naked-PNA condition 10 µM of PNA is added to the gentamicin containing media that the HeLa cells are incubated in for 18 h. Post 18 h of treatment HeLa cells are fixed for imaging or lysed for colony forming unit (CFU) analysis. b Representative images of HeLa cells uninfected (top left), infected and without treatment (bottom left), infected and treated with Naked-PNA (top right), and infected and treated with T3SS-PNA (bottom right). HeLa cells are stained with the nuclear stain DAPI (blue), the membrane stain Phalloidin (pink), and green pixels represent intracellular STm-GFP (green). Images show an evident decrease in STm-GFP during T3SS-α-rpsD or T3SS-α-lexA treatment compared to no treatment or with naked-PNA treatment. c Percent infection (CFU/mL of treatment normalized to no treatment) in the presence or absence of treatment. Significant reduction in STm in HeLa cells when PNA is delivered using the T3SS. Naked-PNA treatment does not produce significant therapeutic effect. d Schematic showing the experimental setup comparing the delivery and release approach to no treatment of an intracellular infection of HeLa cells. The same procedure was followed as in part A with few modifications. Pretreatment of the delivery STm (STm-GFP modified with holin release switch) is done by incubating in either 10 µM of PNA (bottom, T3SS/holin-PNA) or PBS (top, no treatment) for 45 min to allow for PNA uptake. Then HeLa cells are infected with a 4:1 ratio of delivery STm (green) to target STm (red), making the total MOI 10. After removing extracellular bacteria, the infected HeLa cells are incubated for 18 h; during this time the delivery STm releases the PNA to treat the Target STm strain. After 18-h of incubation the HeLa cells are lysed to determine the number of intracellular colony forming units. e CFU ratio of normalized target STm to normalized delivery STm (see methods section for normalization) shows significant reduction compared to no treatment. Triangles represent individual biological replicates of HeLa cells. f Percent infection of target STm (Target STm CFU normalized to no treatment) when using a non-T3SS bacteria ( E. coli MG1655) as the delivery strain shows no reduction ( p > 0.05). g Lysed ESBL KPN (CFU/mL) from intracellular infection in RAW264.7 macrophages at 1 MOI and treated for 18 h with delivery STm at 10 MOI carrying α-rpsD. All data shown are the average of three biological replicates with standard deviation shown as error bars. Grey circles or blue triangles indicate individual biological replicates.

Article Snippet: E. coli MG1655 PNA growth experiments were carried out in M9 media (1x M9 minimal media salts solution (MP Biomedicals), 2.0 mM MgSO 4 , and 0.1 mM CaCl 2 in sterile water) with 0.4% glucose.

Techniques: Infection, Expressing, Incubation, Imaging, Staining, Modification, Standard Deviation

Journal: BMC Microbiology

Article Title: Release of extracellular ATP by bacteria during growth

doi: 10.1186/1471-2180-13-301

Figure Lengend Snippet: ATP supplementation increases the stationary survival of bacteria. E. coli K12, Salmonella enterica Serovar Enteritidis (SE) or Salmonella enterica Serovar Typhimurium (ST) was cultured in M9 minimal medium or M9 minimal medium supplemented with 10 μM or 100 μM of ATP. The rate of survival was determined by comparing bacterial CFU/mL after 7 days of incubation to that after 1 day of incubation. The experiment was performed three times and results are from a representative experiment performed in triplicate. Error bars represent standard deviation. * p < 0.05, Student’s t -test.

Article Snippet: Culture media Luria Bertani (LB) broth and M9 minimal medium were from BD Diagnostics (Sparks, MD).

Techniques: Bacteria, Cell Culture, Incubation, Standard Deviation

Journal: Antibiotics

Article Title: A Pilot Study in Humans on the Urinary Tract Excretion of the FimH Inhibitor 1-Deoxymannose

doi: 10.3390/antibiotics14050498

Figure Lengend Snippet: Utilization of 1-deoxymannose on the growth of Escherichia. coli. Changes in turbidity (600 nm) caused by the growth of Escherichia coli (NBRC 3301) in a M9 minimal medium containing 1% of each sugar (DM, Man, glucose).

Article Snippet: Escherichia coli (NBRC 3301) was shaken at 37 °C for 17 h using 2 mL of M9 minimal medium (DifcoTM M9 Minimal Salt supplemented with MgSO 4 and CaCl 2 , Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing 1% glucose to obtain a culture solution.

Techniques: