TITLE: Compound Screening and Growth Curves
AUTHORS: Carlos Goller

[DESCRIPTION]
Overview
The bacterial genus Delftia was named for the city of Delft in the Netherlands, where the species type was isolated and for the Delft research groups that had a critical role in the early development of bacteriology. Species of Delftia have since been isolated all over the world in different environments and have been known to cause infections in humans.1Delftia are gram-negative rod-shaped aerobes. Species of Delftia are often resistant to several compounds, including the common disinfectant chlorhexidine,2 most antibiotics in the aminoglycoside group,3 and heavy metals.4 One of Delftia’s most surprising talents is its ability to concentrate heavy metals like gold.5 Biofilms found on gold deposits suggest that these bacteria are responsible for their formation.6 Known species of Delftia include D. acidovorans, D. tsuruhatensis, D. deserti, D. lucustris, and D. litopenaei. Refer to Figure 1 for an example of Delftia acidovorans colonies growing on tryptic soy agar (TSA).

 

Where are Delftia spp.found?
Delftia spp. are commonly isolated from aquatic environments such as lakes7 and wastewater8 and are thought to be ubiquitous. Because of their affinity for aquatic environments, Delftia have been identified in organisms and objects frequently found in these environments, such as mosquitoes9 and birds’ feathers.10Delftia aren’t just aquatic microbes, though. These bacteria have been identified in unexpected places like desert soil11 and contact lens cases.12

An Opportunistic Pathogen
Species of Delftia have been known to cause various infections in healthy and immunocompromised people. Many of these infections are (IV) line-related,13,14 but other infections have occurred due to intravenous drug use.3Delftia were also implicated in eye inflammation related to contact lens use.12 NC State students and staff have even found Delftia DNA in surveys of kitchen sinks performed by students in the BIT 477/577 Metagenomics lab module!

Our Project
We will set up a series of high-throughput (HT) assays to test whether different gold chloride concentrations inhibit the growth of different Delftia strains. Gold chloride has been explored for its antibacterial properties15. However, Delftia spp. have a unique non-ribosomal peptide that helps relieve gold toxicity by producing delftibactin5. 
Bacterial strains will be grown in 96-well plates to test multiple concentrations of gold chloride and antibiotics and the effect of miniaturization of the assay. We will set up plates in the first lab session to test several strains at multiple doses with numerous replicates. Plates will incubate for one day at 30ºC before assessing cell viability using a stain for metabolic activity, PrestoBlue®. This approach can be adapted to screen active compounds from a high-throughput screening (HTS) campaign. For this first compound screen, we will focus on the effects of automation and miniaturization on assay reproducibility.

We will test gold chloride concentrations in a twelve-point dose-response curve. At the end of this experiment, you can determine which concentrations are inhibitory by calculating the percent growth inhibition and IC50. We will compare the susceptibility of various Delftia strains.

[BEFORE_START]
Please put on your personal protective equipment (PPE) supplied by your instructors. PPE for this lab includes gloves, disposable lab coats, and eye protection. 

Before you start, clean bench surfaces and pipettors with ethanol. 

[GUIDELINES]
Additional Resource : PrestoBlue Cell Viability Reagent Protocol

[STEPS]
SECTION: Plate Setup and Incubation
1. Read the procedure listed below. Discuss with your lab partner the key steps to program the liquid handler to successfully complete this task. Pay attention to the reagents, tube format, and equipment we have. Remember the goal: reproducibly testing compounds (with replicates) and including negative and positive controls.

SECTION: Plate Setup and Incubation
2.  
SECTION: Plate Setup and Incubation
3. Grow    cultures of Delftia spp. in 2.5 mL of TSB, shaking at   and incubating at 30 °C 

SECTION: Plate Setup and Incubation
4. For each strain, subculture the bacteria to approximately 5 x 105 CFU in 25 mL  of fresh TSB by performing the following:

SECTION: Plate Setup and Incubation
4.1. Measure the OD600 of the overnight culture (1:10 dilution in TSB);
SECTION: Plate Setup and Incubation
4.2. Plug this number into the formula at the bottom of the page and multiply by 10 to ensure we have enough bacteria in each well.
SECTION: Plate Setup and Incubation
4.3. Take the calculated amount (μL) from the overnight culture and add to 25 mL  of fresh tryptic soy broth (TSB); mix well.

SECTION: Plate Setup and Incubation
5. Add 100 µL  of diluted bacterial suspension into each well of rows 2-12.

SECTION: Plate Setup and Incubation
6. Add 194.88 µL  of subcultured bacterial suspension into each well of row 1.

SECTION: Plate Setup and Incubation
7. Add gold chloride (5.12 µL  from a stock solution) to wells B1, C1, and D1.

SECTION: Plate Setup and Incubation
8. Add antibiotic solution (5.12 µL  from a stock solution) to E1, F1, and G1.

SECTION: Plate Setup and Incubation
9. Add water (5.12 µL  of water) to A1 and H1 (to ensure the diluent is not toxic to the bacteria)

SECTION: Plate Setup and Incubation
10. Thoroughly mix the wells in column 1 and remove 100 µL  and add to the wells in column 2.

SECTION: Plate Setup and Incubation
11. Thoroughly mix the wells in column 2 and remove 100 µL  and add to the wells in column 3.

SECTION: Plate Setup and Incubation
12. Continue the serial dilution down the 96-well plate and discard the last 100 μL from column 12 (Note: There should be 100 μL in each well now).
SECTION: Plate Setup and Incubation
13. Incubate at 30 °C  for 1440 min  with    in the LogPhase 600 plate reader. Take A600 readings every 10 min.

SECTION: Plate Setup and Incubation
14. Inspect the 96-well plate for growth. The MIC (Minimum Inhibitory Concentration) is the first concentration at which no growth is observed. (Note: there should be growth in rows A and H).
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15. Read OD600 and add 10 µL  of PrestoBlue to each well.

SECTION: Plate Setup and Incubation
16. Incubate at 30 °C for 30 min . Read OD570 and OD600 for normalization. Review the information provided in the PrestoBlue resource.

SECTION: Plate Setup and Incubation
17. Note the concentration at which each compound inhibits the growth of the bacteria. Record data in your electronic lab notebook. 
 

SECTION: Plate Setup and Incubation
18. Plate Layout
 

 

SECTION: Plate Setup and Incubation
19.