It is estimated that antibiotic-resistant (ABR) infections directly caused 1.27 million deaths and contributed to a further 4.95 million deaths globally in 2019 [1]. In addition, multi-drug resistant infections are increasing as bacteria develop simultaneous resistance mechanisms to numerous antibiotics [2,3]. Despite this mounting threat, no novel class of antibiotics have been introduced into the clinic since the discovery of diarylquinolines in 2004 [4,5]. This ∼20-year innovation gap demonstrates the critical need for novel strategies to combat resistant infections [6,7]. A common first step in the drug development pipeline is the identification of small molecule modulators of a biological target, typically found by screening libraries that cover a wide range of chemical motifs [8]. Given that the hit rates are ∼1 %, screening volume is an important factor [9]. As such, high-throughput screening (HTS) has become an essential element in the antibiotic discovery pipeline and these assays fall into three general categories: in vitro protein assays, reporter fusion read-out methods, and phenotypic assays (Table 1). Herein, we summarize the critical advantages and disadvantages between these strategies both in terms of technology development and information content of the resulting data. We will also highlight recent advancements that applied the principles of HTS to investigate novel bacterial systems or develop innovative approaches to assay design for the discovery of small molecule antibiotic or antivirulence therapeutics.

The most common and straightforward approach to HTS for microbial targets is through the in vitro assessment of purified proteins. These assays typically use fluorescence, luminescence, or colorimetric outputs to identify protein binders or detect the modulation of protein activity. In vitro assays are often more quickly established than other assay types and typically require less time and resources to perform in high-throughput. Another common HTS method in bacteria is the use of reporter fusion strains that enable monitoring of the expression of key genes involved in a pathway of interest. These assays require fusion of the promoter of a gene or other biosensor of interest to an unrelated reporter gene whose expression can be monitored using a fluorescent, luminescent, or colorimetric output. The final category of assay that we will discuss is cellular assays, which are essential for understanding the overall effects of a given molecule on therapeutically relevant phenotypes (e.g., cell death). While these screens provide invaluable leads, they often lack information about the targets of the identified molecules and can be the most difficult to perform as a HTS.