微生物学

Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen–host interactions in filamentous fungi.

Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as Mycobacterium tuberculosis (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1–4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1–4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS).

Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial–drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs.

Key features

• Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant.

• Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine.

• A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate.

• The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.

Graphical overview

Protocol overview. (1) Bacteria are incubated with gemcitabine for a set period of time. (2) Samples are removed from co-incubated suspensions and filtered to remove bacteria to halt gemcitabine degradation. (3) A gemcitabine-sensitive reporter strain is then added to the conditioned supernatant and is supplemented with growth media. (4) Growth of the reporter strain is monitored over time. (5) Results from the growth experiments are used to infer the concentration of gemcitabine in the co-culture supernatant and the drug degradation rate.

Bacteriocins are antimicrobial peptides with activity against antibiotic resistant bacterial pathogens. Here, we describe a set of methods aimed at purifying, identifying, and characterizing new bacteriocins. The purification consists of ammonium sulphate precipitation, cation-exchange chromatography, and reversed-phase chromatography. The yield of the bacteriocin is quantified by bacteriocin antimicrobial activity in a microtiter plate assay after each purification step. The mass of the purified bacteriocin is assessed by MALDI TOF MS analysis of the active fractions after reversed-phase chromatography. The mass is compared with the theoretical mass based on genetic information from the whole genome sequencing of the bacteriocin producer strain. Physicochemical characterization is performed by assessing antimicrobial activity following heat and protease treatments. Fluorescent techniques are used to examine the capacity of the bacteriocin to disrupt membrane integrity. Herein a set of protocols for purification and characterization of the bacteriocin nisin Z is used as a typical example in this paper.

Bacterial studies based on growth curves are common in microbiology and related fields. Compared to the standard photometer and cuvette based protocols, bacterial growth curve measurements with microplate readers provide better temporal resolution, higher efficiency, and are less laborious, while analysis and interpretation of the microplate-based measurements are less straightforward. Recently, we developed a new analysis method for evaluating bacterial growth with microplate readers based on time derivatives. Here, we describe a detailed protocol for this development and provide the homemade program for the new analysis method.

Coronaviruses are important human pathogens, among which the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. To combat the SARS-CoV-2 pandemic, there is a pressing need for antivirals, especially broad-spectrum antivirals that are active against all seven human coronaviruses (HCoVs). For this reason, we are interested in developing antiviral assays to expedite the drug discovery process. Here, we provide the detailed protocol for the cytopathic effect (CPE) assay and the plaque assay for human coronaviruses 229E (HCoV-229E), HCoV-OC43, and HCoV-NL63, to identify novel antivirals against HCoVs. Neutral red was used in the CPE assay, as it is relatively inexpensive and more sensitive than other reagents. Multiple parameters including multiplicity of infection, incubation time and temperature, and staining conditions have been optimized for CPE and plaque assays for HCoV-229E in MRC-5, Huh-7, and RD cell lines; HCoV-OC43 in RD, MRC-5, and BSC-1 cell lines, and HCoV-NL63 in Vero E6, Huh-7, MRC-5, and RD cell lines. Both CPE and plaque assays have been calibrated with the positive control compounds remdesivir and GC-376. Both CPE and plaque assays have high sensitivity, excellent reproducibility, and are cost-effective. The protocols described herein can be used as surrogate assays in the biosafety level 2 facility to identify entry inhibitors and protease inhibitors for SARS-CoV-2, as HCoV-NL63 also uses ACE2 as the receptor for cell entry, and the main proteases of HCoV-OC43 and SARS-CoV-2 are highly conserved. In addition, these assays can also be used as secondary assays to profile the broad-spectrum antiviral activity of existing SARS-CoV-2 drug candidates.

Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite’s liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype.

Graphical abstract:

Overview of P. vivax liver stage screening assay performed at the Institute Pasteur of Cambodia.

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions.

Graphic abstract:

Schematic representation of the ODELAM platform