Kevin Wyllie Week 11

Milestones This Week

1. Understand experimental design.
2. Establish sample-data relationship
   • raw.zip and .sdrf.txt files
   • Construct sample-data flowchart/diagram
3. If possible develop compiled raw data file

10 Unfamiliar Terms

1. sessile cells
2. planktonic cells
3. anaplerotic pathway
4. sonication
5. RT-PCR
6. flow cytometry
7. exconjugants

Article Outline

Significance of this Study

• Burkholderia cenocepacia is a a member of the Burkholderia cepacia complex (Bcc), which is comprised of 17 similar species.
• B. cencocepacia is an opportunistic pathogen, which causes severe lung infection in cystic fibrosis patients, as well as those who are generally immunocompromised.
• Infections with Bcc organisms are difficult to eliminate for three reasons:
  • Widespread antibiotic resistance.
  • Ability to form biofilms (forming a physical barrier from antibiotics).
  • "Persister cells," which are a phenotypic variant exhibiting even greater antibiotic resistance. Though they comprise a very small fraction of a population, persister cells can linger during/after antibiotic treatment, and can then repopulate when the antibiotic is removed.
• One of the proposed mechanisms by which antimicrobials work is overstimulation of NADH oxidation, hyperactivating the electron transport chain. This releases large amounts of reactive oxygen species (ROS), which damage proteins containing iron-sulfur clusters, releasing ferrous iron. Finally, this ferrous iron damages DNA, lipids and proteins, leading to eventual cell death.
• The glyoxylate cycle can be induced in many microorganisms to lessen the generation of ROS, as it skips two of the steps in the TCA cycle which would normally product NADH. This may be a primary mechanism of antibiotic resistance in Bcc (also mentioned are the ubiquitous drug efflux pumps and beta-lactamases).
  • A prior study (Van Schaik et al.) found that inhibition of isocitrate lyase - the enzyme which initiates the shunting of TCA into the glyoxylate cycle - forced Burkholderia pseudomallei back into a state of antibiotic susceptibility, supporting this hypothesis.
• This study investigates:
  • Levels of persister cells in Bcc biofilms.
  • The molecular root of antibiotic resistance.
  • Effective methods for eradicating Bcc biofilms.

Methods

Minimal Inhibitory Concentration (MIC)

• Minimal Inhibitory Concentrations (MIC) were determined, using the EUCAST broth microdilution protocol, for the following antimicrobials:
  • Tobramycin
  • Ciproflaxin
  • Itaconate with 3-nitropropionate
  • 2-thenoyltrifluoroacetone
• This was done in with duplicates, in 96-well microtiter plates, treated and incubated for 24 hours.
• MIC's were defined as the lowest concentration at which no significant difference in OD590 was observed, between blank and inoculated wells.

Quantification of Persister Cells in Biofilms and Planktonic Cultures

• Biofilm and planktonic cultures were exposed to tobramycin and ciproflaxin and the numbers of surviving cells were determined.
• Cultures were grown for 24 hours, and then exposed to either antibiotic (or a physiological saline control) for another 24 hours.
  • For biofilm cultures:
    • Cultures were grown in 96-well microtiter plates. Twelve wells were used for each condition, but it appears that not all were quantified (see below).
    • Both incubation periods were at 37°C.
    • Cells were harvested and plated on LB media for quantification. All experiments had two or more replicates.
  • For planktonic cultures:
    • Cultures were grown overnight in a shaking warm water bath. After 24 hours, cell suspensions with OD590's of 1 were centrifuged in falcon tubes, and then resuspended in either antibiotic or the control.
    • Cells were resuspended in physiological saline and quantified by plating on LB media.

RNA Extraction and Microarray Analysis

• Biofilms of B. cenocepacia J2315 were grown in 96-well microtiter plates for 24 hours, and then incubated in the presence of either tobramycin (4x MIC) or a saline control solution for another 24 hours.
  • Cells were collected by vortexing and sonication.
  • RNA was:
    • Extracted with the Ambion RiboPure Bacteria Kit.
    • Treated with DNase I for 1 hour.
    • Concentrated with Microcon YM-50 filters.
    • Linearly amplified with the Ambion MessageAmp II-Bacteria Kit.
• cDNA was synthesized and hybridized to custom made 4x44K Agilent arrays.
• GeneSpring GX 7.3 was used for array analysis.
• Data was normalized using Agilent's recommended procedure for two-color micorarrays, and a T-test was performed.

Note: Although neither the methods nor results sections mention the amount of replicates used, the .sdrf file indicates that five replicates were used for untreated samples, and three replicates were used for the tobramycin treated samples. The author has also stated that the reference sample for each array was the organism's genomic DNA.

Quantitative RT-PCR

• 11 genes were selected to be quantified, as a means of validating the microarray results.
• Treated and untreated biofilms were grown as described in the microarray analysis methods.
  • RNA was extracted, and cDNA was was synthesized with BioRad's iScript cDNA Synthesis Kit.
• Forward and reverse primers were designed using tools on NCBI's website, and BLAST was used (with B. cenocepacia J2315's genome) to verify the specificity of these primers.
• Samples were spotted in duplicate, along with a control, containing no cDNA.
• BioRad's CFX96 Real-Time System C1000 Thermal Cycler was used.
• A melting curve was generated at the end of each run.
• Three "reference genes" were used to normalize the data. An algorithm called GeNorm was used to confirm a stable expression for these three genes.

Flow Cytometry

• After being resuspended in physiological saline, both treated and untreated biofilms (again, both prepared as described in the microarray methods) were stained with an ROS-specific dye, and the passed through a Cyan ADP flow cytometer for quantification.
  • Biofilms were dyed by incubating for 30 minutes in the dark, in the presence of the dye.
• Data for at least 50,000 cells was collected in each sample.

Inhibition of SOD, ICL and SDH

• To evaluate the importance of superoxide dismutase (SOD), isocitrate lyase (ICL) and succinate dehydrogenase (SDH), biofilm persister cells (presumably prepared as described above, though not explicitly stated in the article) were quantified after exposure to 4x MIC tobramycin in conjunction with the following inhibitory agents:
  • Diethyldithiocarbamate, inhibiting SOD.
  • Itaconate or 3-NP, both inhibiting ICL.
  • 2-thenoyltrifluoracetone, inhibiting SDH.
• Quantification was presumably done by flow cytometry, however the exact method is not mentioned.

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