Vpachec3 Week 11

10 Biological Terms and Definitions

1. Aminoglycoside antibiotic- group of antibiotics active against many acrobic Gram-negative and some Gram-positive bacteria. Composed of two or more amino sugars attached by a glycosidic linkage to a hexose nucleus; polycationic and highly polar compounds. Inhibit bacterial protein synthesis by binding to a site on the 30S ribosomal subunit thereby altering codon-anticodon recognition.
2. Fluoroquinolone- bactericidal and inhibit the activity of DNA gyrase. Newer fluoroquinolones (e.g. ciprofloxacin) had a broader spectrum of activity
3. Sessile cells- (1)of, pertaining to, or relating to the state of sessility or the inability to move actively or spontaneously.(2) being permanently attached to the substrate or to the base, hence, not freely moving[1]
4. cDNA-Complementary DNA Viral reverse transcriptase can be used to synthesize DNA that is complementary to RNA. The cDNA can be used, for example, as a probe to locate the gene or can be cloned in the double-stranded form.
5. Flow cytometry-Slightly imprecise but common term for the use of the fluorescence activated cell sorter (FACS). Cells are labelled with fluorescent dye and then passed, in suspending medium, through a narrow dropping nozzle so that each cell is in a small droplet. A laser-based detector system is used to excite fluorescence and droplets with positively fluorescent cells are given an electric charge. Charged and uncharged droplets are separated as they fall between changed plates and so collect in different tubes. the machine can be used either as an analytical tool, counting the number of labelled cells in a population, or to separate the cells for subsequent growth of the selected population. further sophistication can be built into the system by using a second laser system at right angles to the first look at a sec on fluorescent label or to gauge cell size on the basis of light scatter. The great strength of the system is that it looks at larger numbers of individual cells and makes possible the separation of populations with, for example, particular surface properties.
6. Isocitrate- an intermediate in the tricarboxylic acid cycle (citric acid cycle)
7. Persister cells- bacterial cells that survive killing by antibiotics that block synthesis of peptidoglycan or DNA, but remain sensitive to that antibiotic upon being regrown and give rise to the same small fraction of persisters. [2]
8. Exconjugants- a member of a conjugating pair of protozoan ciliates after separation and prior to the subsequent mitotic division of each of the exconjugant's.[3]
9. Mutagenesis- act of mutation by deleting or changing the nucleotide sequence[4]
10. Glyoxylate shunt- (See glyoxalate cycle) Glyoxylate cycle: metabolic pathway present in bacteria and in the glyoxisome of plants, in which two acetyl-CoA molecules are converted to a 4-carbon dicarboxylic acid, initially succinate. Includes two enzymes not found elsewhere, isocitrate lyase and malate synthase. Permits nest synthesis of carbohydrates from lipid, and hence is prominent in those seeds in which lipid is the principle food reserve.

Definitions for numbers 1, 2, 4, 5,6 and 10 were found in Dictionary of Cell and Molecular Biology (Citation below).

Lackie, J. M. (2007). Dictionary of Cell and Molecular Biology. Kidlington, GBR: Academic Press. Retrieved from http://www.ebrary.com

Article Outline

1. Introduction
   • • Bcc are opportunistic pathogens
     • found in cystic fibrosis patients
     • Infections are species dependent
     • Hard to eradicate because they are good at resisting antibiotics and are good at creating biofilms
2. Materials and Methods
   • Strains and Culture Conditions
     • 18 strains in the Burkholderia cepacia group
     • Plated on agar plates at 37 degrees Celsius over night
     • Cultures diluted in broth and incubated at 37 degrees Celsius
   • Minimal Inhibitory Concentration (MIC)
     • Used EUCAST broth microdiluation protocol to find MICs
     • Tested:Tobramycin and Ciprofloxacin,Itaconate and 3-nitropropionate,2-Thenoyltrifluoroacetone
     • Performed in duplicate and replicates
     • Never differed more than 2 fold
   • Quantification of Persister Cells in Biofilms and Planktonic Cultures
     • Aim: to see how many surviving persister cells there are
     • Day old biofilms and planktonic cultures were treated with tobramycin and ciprofloxacin
     • Used 96 well micro titer plate for growth
     • After 24 hrs, the supernatant was removed and put into antibiotic solution mixed in physiological saline
     • Cells were harvested and plated onto agar plates
     • Planktonic cultures needed extra preparation to reach optical density of 1
   • RNA Extraction and Microarray Analysis
     • Biofilms exposed to either antibiotic or 0.9% salt solution
     • After 24hr, the sessile cells were prepped to take out the RNA
     • Right before microarray analysis, the samples were concentrated and amplified
     • Gene expression analysis was executed
     • Data used 2 color Agilent microarrays
     • T-test analysis was used
   • Quantitative RT-PCR
     • Chose 11 selected genes to use RT-qPCR on
     • RNA was extracted from biofilms
     • cDNA was synthesized
     • forward and reverse primers were chose through genome comparison in BLAST
     • the control had no cDNA
     • 3min denaturation followed by 40 amphilication cycles
   • Flow Cytometry
     • Aim: to see induction of reactive oxygen species by tobramycin
     • Stained biofilms with ROS-specific dyes
     • Dye sits for 30 mins in the dark
     • Rinsed off and physiological saline was added in well
     • Cells harvested and resuspended in diluted physiological saline
     • Cells were measured using flow cytometer
     • 50,000 cells per sample
   • Effect of SOD,ICL or SDH Inhibition on Survival of Bcc Persisters
     • surviving cells treated with tobramycin and SOD inhibitor diethyldithiocarbamate --> Importance of protection against ROS
     • biofilms treated an duntreated with itaconate or 3-NP (ICL inhibitors) --> importance of ICL persistence
     • surviving cells treated with 2-thenoyltrifluoroacetone
   • Construction of ICL Mutant
     • Aim: creation of unmarked nonpolar gene deletion
     • Deletion of BCAL2118 and BCAM1588 (ICL encoding )
     • Created double mutant
     • Insertion of mutagenesis plasmid (pGPI-SceI-XCm) for tetracycline-resistant and Tp-susceptible
     • Insertion confirmed through PCR
   • Construction of SDH Antisense Overexpression Mutants
     • Antisense overexpression mutant involved amplifying BCAM0967 and BCAM0968
     • Needed to confirm the significance of SDH
3. Results and Discussion
   • • TCA cycle was downregulated
     • Gene expression in the electron transport chain was downregulated
     • as a result, avoids the production of ROS
     • Genes in the production of NADPH were upregulated
     • Glyoxylate shunt and ICL encoding genes were upregulated
     • Less persister cells survived when in the presence of ICL and SDH
     • Overall:mechanism is widespread throughout the various species new approaches to the treatment of persister-related infections

Figures and Tables

• Figure 1: percentage of cells which survived treatment of tobramycin and ciproflaxin. Open circles are biofilms and triangles are planktonic cultures
• Figure 2: fluorescent signal for tobramycin-treated and untreated biofilms. The differences between the two graphs are statistically significant
• Figure 3: percentage of surviving cells in C5424 and two mutants (MDL1 and MDL2) after treated with tobramycin
• Figure 4: percentage of surviving cells in B.cenocepacia J2315 with either only tobramycin (control) or tobramycin and diethlydithiocarbamate
• Figure 5: percentage of surviving cells in J2315 after treated with tobramycin or with ciprofloxacin, both supplemented with itaconate or 3-NP
• Figure 6: percentage of surviving cells after treatment with tobramycin or combo of tobramycin and itaconate
• Figure 7A: percentage of surviving cells for the double mutant, double mutant with 3-NP, the wildtype and the wildtype with 3-NP
• Figure 7B: percentage of surviving cells double mutant strands treated with tobramycin and the double mutant supplemented with 2-thenoyltrifluoroacetone
• Table 5: shows the difference in gene expression as fold changes in the microarray analysis and the qPCR experiment. It shows glyoxylate shunt, Tricarboxylic acid cycle, oxidative phosphorylation, NAD(P)H production and response to oxidative stress.

Previous Studies

Most of the studies referenced in the paper were for methodologies of the different experiments run. Toward the conclusion we see that there is an article where they "reawaken" persister cells to counteract the phenotypic changes which allow for antimicrobial resistance. While this study, we see a focus on targeting the cell's mechanisms to deal with ROS. It's more of a proactive approach rather than the reactive approach in the prior literature.

Links

Vpachec3 User Page

GÉNialOMICS Links

Weekly Group Assignments	Shared Group Journals	Project Links	Team Members
Week 10 Week 11 Week 12 Week 14 Week 15	GÉNialOMICS Week 10 GÉNialOMICS Week 11 GÉNialOMICS Week 12 GÉNialOMICS Week 14 GÉNialOMICS Week 15	GENialOMICS Home Page GENialOMICS Deliverables Gene Database Project Overview Instructions for Project Deliverables Project Manager Guild Page GenMAPP User Guild Page Quality Assurance Guild Page Coder Guild Page	Project Manager/Coder: Anu Varshneya Quality Assurance: Brandon Litvak GenMAPP User: Veronica Pacheco GenMAPP User: Kevin Wyllie