High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Summary

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Abstract

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

Introduction

Bacterial cultures contain a small subpopulation of persister cells that are temporarily tolerant to unusually high levels of antibiotics. Persister cells are genetically identical to their antibiotic-sensitive kins, and their survival has been attributed to transient growth inhibition¹. Persister cells were first discovered by Gladys Hobby² but the term was first used by Joseph Bigger when he identified them in penicillin-treated Staphylococcus pyogenes cultures³. A seminal study published by Balaban et al.⁴ discovered two persister types: type I variants that are....

Protocol

1. Preparation of growth medium, ofloxacin solution and E. coli cell stocks

1. Regular Luria-Bertani (LB) medium: Add 10 g/L of tryptone, 10 g/L of sodium chloride (NaCl) and 5 g/L of yeast extract in deionized (DI) water. Sterilize the medium by autoclaving.
2. LB agar plates: Add 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract and 15 g/L agar in DI water and sterilize the medium by autoclaving. At the desired temperature (~55 °C), pour ~30 mL of agar medium into square plates (10 x 10 cm). Dry the plates and store at 4 °C.
3. Modified LB medium: Add 10 g/L of tryptone and 5 g/L of yeast extract in....

Discussion

The high throughput persister assay described here was developed to elucidate the effects of various chemicals on E. coli persistence. In addition to commercial PM plates, microarrays can be constructed manually as described in step 4.2. Moreover, the protocol presented here is flexible and can be used to screen other microarrays, such as drug panels and cell libraries, that are in 96 well plate formats. The experimental conditions including the growth phase, inoculation rate and medium can be adjusted to test t.......

Materials

Name	Company	Catalog Number	Comments
14-ml test tube	Fisher Scientific	14-959-1B
E. coli strain MG1655	Princeton University		Obtained from Brynildsen lab
Flat-bottom 96-well plate	USA Scientific	5665-5161
Gas permeable sealing membrane	VWR	102097-058	Sterilized by gamma irradiation and free of cytotoxins
Half-area flat-bottom 96-well plate	VWR	82050-062
LB agar	Fisher Scientific	BP1425-2	Molecular genetics grade
Ofloxacin salt	VWR	103466-232	HPLC ≥97.5
Phenotype microarray (PM-9 and PM-10)	Biolog	N/A	PM-9 and PM-10 plates contained various osmolytes and buffers respectively
Round-bottom 96-well plate	USA Scientific	5665-0161
Sodium chloride	Fisher Scientific	S271-500	Certified ACS grade
Sodium nitrate	Fisher Scientific	AC424345000	ACS reagent grade
Sodium nitrite	Fisher Scientific	AAA186680B	98% purity
Square petri dish	Fisher Scientific	FB0875711A
Tryptone	Fisher Scientific	BP1421-500	Molecular genetics grade
Varioskan lux multi mode microplate reader	Thermo Fisher Scientific	VLBL00D0	Used for optical density measurement at 600 nm
Yeast extract	Fisher Scientific	BP1422-100	Molecular genetics grade