Team:Paris Bettencourt/Testing

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Testing

StarCore constructs were tested for their efficacy by determining their minimum inhibitory concentration (MIC) and growth curve assay compared to established an antibiotic and antimicrobial peptide (AMP) namely, Ampicillin and Ovispirin (OV-1), respectively. In addition, the mechanism of action for both the controls (Ampicillin and Ovispirin/ OV-1) and Starcores were shown through a time-lapse video taken from the agar pad experiment. The following below are the in-depth description of each of the experiments mentioned highlighting their principles, utilities, and, specificities.

Minimum inhibitory concentration (MIC) determination

The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after 16 hours of incubation (Andrews et. al., 2001). In order to investigate the efficiency of our StarCores and compare them to established antibiotics and antimicrobial peptides (AMPs), we performed MIC determinations. First, we built our experimental protocol for the two positive controls, Ampicillin and OV-1, expected to yield MICs in a range consistent of previous reports (Andrews et. al. 2001; Lam et. al. 2016). Overnight cultures of tested bacteria (E. coli or B. subtilis) are diluted 40X into fresh media and allowed to grow up to log phase (2 hours) at 37°C with agitation of 220 RPM. After washing them 3 times with 1 mL of PBS, we resuspend the bacteria in LB to OD600 ~ 0.01. A droplet of bacteria is seeded into Mueller Hinton Broth (MHB) media, supplied by serial dilution with various concentration of antimicrobial to be tested in a 96-well plate. The plate was incubated at 37°C for 20 - 24 hours with agitation. Afterwards, we measure the bacterial survival under each condition by measuring the OD600 in a spectrophotometer. The smallest concentration of antimicrobial for which there was no bacterial growth is considered to be the MIC of the corresponding antimicrobial.

Figure 1:Diagram of the Minimum Inhibitory Concentration Protocol.

Growth curve deficiency measurement

In order to understand the impact of the antimicrobials on the growth of E. coli and B. subtilisthe growth curve in the presence of these compounds was determined (Lam et. al., 2016; Cao et. al., 2018). The bacterial seed culture is prepared as described for the t MIC determination in 96 well plates. Then, we measured the OD600 with a kinetic interval of 15 minutes for 12 hours with agitation.

Figure 2: Diagram for the Growth Curve Protocol.

Agar pad method and time-lapse microscopy

Agar pads consists of a thin layer of 1.5% agarose in which bacterial cells are dispensed on the surface. A glass coverslip is place on top to seal the microscopic specimen (Joyce et. al., 2001). We then image the bacteria on agar pad via time-lapse microscopy, providing us with information regarding the impact of an antimicrobial on bacterial physiology (Jong et. al., 2011). The tested bacteria is collected from log phase, washed, and then dispensed on agar pads containing a certain antimicrobial concentration. We made two different agar pads: one without antimicrobial (negative control) and another with 1X MIC of the antimicrobial. Five microscopic field positions were imaged overnight. A time-lapse movie were made from the images taken using ImageJ.

Figure 3: Diagram for the Agar pad preparation.

References:

1) Lam, S.J., O’ Brien-Simpson, N.M., Pantarat, N., Sulistio, A., Wong, E.H.H., Chen, Y., Lenzo, J.C., Holden, J.A., Blencowe, A., Reynolds, E.C., and Qiao, G.G. (2016). Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers. Nature Microbiology 1(11):16162. 2) Andrews, J. (2002). Determination of minimum inhibitory concentrations. J Antimicrob Chemother 49(6):1049. 3) Cao, J., de la Fuente-Nunez, C., Ou, R.W., Torres, M.D.T., Pande, S.G., Sinskey, A.J., and Lu, T.K. (2018). Yeast-based Synthetic Biology Platform for Antimicrobial Peptide Production. ACS Synth. Biol. 7, 896 - 902. 4) Graham, J., Robertson, B.D., and Williams, K.J. (2011). A modified agar pad method for mycobacterial live-cell imaging. BMC Research Notes 4:73.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
paris-bettencourt-2018@cri-paris.org