Antimicrobial susceptibility testing plays a pivotal role in the discovery of new antibiotics. However, the development of simple, sensitive, and rapid assessment approaches remains challenging. Herein, we report an activated alkyne-based cascade signal amplification strategy for ultrafast and high-throughput antibiotic screening. First of all, a novel water-soluble aggregation-induced emission (AIE) luminogen is synthesized, which contains an activated alkyne group to enable fluorescence turn-on and metal-free click bioconjugation under physiological conditions. Taking advantage of the in-house established method for bacterial lysis, a number of clickable biological substances (i.e., bacterial solutes and debris) are released from the bacterial bodies, which remarkably increases the quantity of analytes. By means of the activated alkyne-mediated turn-on click bioconjugation, the system fluorescence signal is significantly amplified due to the increased labeling sites as well as the AIE effect. Such a cascade signal amplification strategy efficiently improves the detection sensitivity and thus enables ultrafast antimicrobial susceptibility assessment. By integration with a microplate reader, this approach is further applied to high-throughput antibiotic screening.

A recently-validated and underexplored drug target in Mycobacterium tuberculosis is PptT, an essential phosphopantetheinyl transferase (PPTase) that plays a critical role in activating enzymes for both primary and secondary metabolism. PptT possesses a deep binding pocket that does not readily accept labelled coenzyme A analogues that have previously been used to screen for PPTase inhibitors. Here we report on the development of a high throughput, colourimetric screen that monitors the PptT-mediated activation of the non-ribosomal peptide synthetase BpsA to a blue pigment (indigoidine) synthesising form in vitro. This screen uses unadulterated coenzyme A, avoiding analogues that may interfere with inhibitor binding, and requires only a single-endpoint measurement. We benchmark the screen using the well-characterised Library of Pharmaceutically Active Compounds (LOPAC1280) collection and show that it is both sensitive and able to distinguish weak from strong inhibitors. We further show that the BpsA assay can be applied to quantify the level of inhibition and generate consistent EC50 data. We anticipate these tools will facilitate both the screening of established chemical collections to identify new anti-mycobacterial drug leads and to guide the exploration of structure-activity landscapes to improve existing PPTase inhibitors.

The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living Escherichia coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. A random forest analysis of in vitro data revealed molecular mass, minimum projection area, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our dataset of 27 antibiotics from different structural classes to reported MIC values of nine clinically relevant pathogens allowed to distinguish active from nonactive compounds based on their low in vitro permeability specifically for Gram-negatives. The model may help to identify poorly permeable antimicrobial candidates before testing them on living bacteria.

Bacterial type II phosphopantetheinyl transferases (PPTases), required for the activation of many cellular mega-synthases, have been validated as promising drug targets in several pathogens. Activation of the blue-pigment-synthesizing nonribosomal peptide synthetase BpsA by a target PPTase can be used to screen in vitro for new antibiotic candidates from chemical libraries. For a complete screening platform, there is a need to also counter-screen inhibitors for cross-reactivity with the endogenous human Type II PPTase (hPPTase), as this is a likely source of toxicity. As hPPTase is unable to recognize the PCP-domain of native BpsA, we used a combination of directed evolution and rational engineering to generate a triple-substitution variant that is able to be efficiently activated by hPPTase. Our engineered BpsA variant was able to readily detect inhibition of both hPPTase and the equivalent rat PPTase by broad-spectrum PPTase inhibitors, demonstrating its potential for high-throughput counter-screening of novel antibiotic candidates.

Droplet microfluidics has recently evolved as a prominent platform for high-throughput experimentation for various research fields including microbiology. Key features of droplet microfluidics, like compartmentalization, miniaturization, and parallelization, have enabled many possibilities for microbiology including cultivation of microorganisms at a single-cell level, study of microbial interactions in a community, detection and analysis of microbial products, and screening of extensive microbial libraries with ultrahigh-throughput and minimal reagent consumptions. In this book chapter, we present several aspects and applications of droplet microfluidics for its implementation in various fields of microbial biotechnology. Recent advances in the cultivation of microorganisms in droplets including methods for isolation and domestication of rare microbes are reviewed. Similarly, a comparison of different detection and analysis techniques for microbial activities is summarized. Finally, several microbial applications are discussed with a focus on exploring new antimicrobials and high-throughput enzyme activity screening. We aim to highlight the advantages, limitations, and current developments in droplet microfluidics for microbial biotechnology while envisioning its enormous potential applications in the future.

　A useful tool for the screening of fungi producing biologically active secondary metabolites such as antibiotics and cytotoxic substances has been developed. An agar plate-organic solvent interface cultivation (A/S-IFC) system, which comprised a hydrophobic organic solvent (upper phase) , a fungal mat (middle phase) and an agar plate (lower phase) , was constructed. The metabolite profiles were compared among the A/S-IFC, a traditional submerged cultivation (SmC) and an extractive liquid surface immobilization (Ext-LSI) system consisted of a hydrophobic solvent (upper phase) , a fungal cells-ballooned microspheres (middle phase) and a liquid medium (lower phase) , with high-performance liquid chromatography-photodiode array detector (HPLC-PDA) . In the A/S-IFC, many hydrophobic metabolites vastly different from those in the SmC were accumulated in the organic phase as with the Ext-LSI. For example, a valuable azaphilone, sclerotiorin, was remarkably produced into the organic phase in the A/S-IFC. The A/S-IFC was applied to the screening of antibiotic-producing fungi. As a result of paper disk method, it was found that 321 isolated among 811 strains produced antifungal metabolites (hit rate, 39.6%) . Furthermore, 8, 23, and 30 strains also produced cytotoxic metabolites against SKOV-3 (human ovary adenocarcinoma) , MESO-1 (human malignant pleural mesothelioma) , and Jurkat cells (immortalized human T lymphocyte) .

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

This essay details a historical crossroad in biochemistry and microbiology in which penicillin was a co-agent. I narrate the trajectory of the bacterial cell wall as the precise target for antibiotic action. As a strategic object of research, the bacterial cell wall remained at the core of experimental practices, scientific narratives and research funding appeals throughout the antibiotic era. The research laboratory was dedicated to the search for new antibiotics while remaining the site at which the mode of action of this new substance was investigated. This combination of circumstances made the bacterial wall an ontology in transit. As invisible as the bacterial wall was for clinical purposes, in the biological laboratory, cellular meaning in regard to the action of penicillin made the bacterial wall visible within both microbiology and biochemistry. As a border to be crossed, some components of the bacterial cell wall and the biochemical destruction produced by penicillin became known during the 1950s and 1960s. The cell wall was constructed piece by piece in a transatlantic circulation of methods, names, and images of the shape of the wall itself. From 1955 onwards, microbiologists and biochemists mobilized new names and associated conceptual meanings. The composition of this thin and rigid layer would account for its shape, growth and destruction. This paper presents a history of biochemical morphology: a chemistry of shape - the shape of bacteria, as provided by its wall - that accounted for biology, for life itself. While penicillin was being established as an industrially-manufactured object, it remained a scientific tool within the research laboratory, contributing to the circulation of further scientific objects.