Microbiology

Chemotaxis refers to the ability of organisms to detect chemical gradients and bias their motion accordingly. Quantifying this bias is critical for many applications and requires a device that can generate and maintain a constant concentration field over a long period allowing for the observation of bacterial responses. In 2010, a method was introduced that combines microfluidics and hydrogel to facilitate the diffusion of chemical species and to set a linear gradient in a bacterial suspension in the absence of liquid flow. The device consists of three closely parallel channels, with the two outermost channels containing chemical species at varying concentrations, forming a uniform, stationary, and controlled gradient between them. Bacteria positioned in the central channel respond to this gradient by accumulating toward the high chemoattractant concentrations. Video-imaging of bacteria in fluorescent microscopy followed by trajectory analysis provide access to the key diffusive and chemotactic parameters of motility for the studied bacterial species. This technique offers a significant advantage over other microfluidic techniques as it enables observations in a stationary gradient. Here, we outline a modified and improved protocol that allows for the renewal of the bacterial population, modification of the chemical environment, and the performance of new measurements using the same chip. To demonstrate its efficacy, the protocol was used to measure the response of a strain of Escherichia coli to gradients of α-methyl-aspartate across the entire response range of the bacteria and for different gradients.

Genome walking, a molecular technique for mining unknown flanking DNAs, has a wide range of uses in life sciences and related areas. Herein, a simple but reliable genome walking protocol named primer extension refractory PCR (PER-PCR) is detailed. This PER-PCR-based protocol uses a set of three walking primers (WPs): primary WP (PWP), secondary WP (SWP), and tertiary WP (TWP). The 15 nt middle region of PWP overlaps the 3' region of SWP/TWP. The 5' regions of the three WPs are completely different from each other. In the low annealing temperature cycle of secondary or tertiary PER-PCR, the short overlap mediates the annealing of the WP to the previous WP site, thus producing a series of single-stranded DNAs (ssDNA). However, the 5' mismatch between the two WPs prevents the template DNA from synthesizing the WP complement at its 3' end. In the next high annealing temperature cycles, the target ssDNA is exponentially amplified because it is defined by both the WP and sequence-specific primer, while non-target ssDNA cannot be amplified as it lacks a binding site for at least one of the primers. Finally, the target DNA becomes the main PER-PCR product. This protocol has been validated by walking two selected genes.

Wastewater-based surveillance (WBS) can provide a wealth of information regarding the health status of communities from measurements of nucleic acids found in wastewater. Processing workflows for WBS typically include sample collection, a primary concentration step, and lysis of the microbes to release nucleic acids, followed by nucleic acid purification and molecular-based quantification. This manuscript provides workflows from beginning to end with an emphasis on filtration-based concentration approaches coupled with specific lysis and nucleic acid extraction processes. Here, two WBS processing approaches are presented, one focusing on RNA-specific pathogens and the other focused on DNA-specific pathogens found within wastewater: 1) The RNA-specific approach, employed for analyzing RNA viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) couples electronegative filtration of wastewater with the placement of the filter within a lysis buffer followed by direct RNA extraction. 2) The DNA-specific approach, employed for analyzing DNA pathogens like Candida auris, uses size selection membranes during filtration, subsequently followed by a lysis buffer, bead-beating, and DNA extraction. Separate workflows for RNA versus DNA isolations have the advantage of improving the detection of the target pathogen. A novel aspect of the RNA-specific workflow is the direct extraction of nucleic acids from filter lysates, which shows enhanced recoveries, whereas the DNA-specific approach requires bead beating prior to extraction. Novelty is also provided in a new qPCR approach called Volcano 2nd Generation (V2G), which uses a polymerase capable of using RNA as a template, bypassing the reverse transcriptase step normally required for qPCR.

Campylobacter jejuni, a widespread pathogen found in birds and mammals, poses a significant risk for zoonosis worldwide despite its susceptibility to environmental and food-processing stressors. One of its main survival mechanisms is the formation of biofilms that can withstand various food-processing stressors, which is why efficient methods for assessing biofilms are crucial. Existing methods, including the classical culture-based plate counting method, biomass-staining methods (e.g., crystal violet and safranin), DNA-staining methods, those that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts and resazurin), immunofluorescence with flow cytometry or fluorescence microscopy, and PCR-based methods for quantification of bacterial DNA, are diverse but often lack specificity, sensitivity, and suitability. In response to these limitations, we propose an innovative approach using NanoLuc as a reporter protein. The established protocol involves growing biofilms in microtiter plates, washing unattached cells, adding Nano-Glo luciferase substrate, and measuring bioluminescence. The bacterial concentrations in the biofilms are calculated by linear regression based on the calibration curve generated with known cell concentrations. The NanoLuc protein offers a number of advantages, such as its small size, temperature stability, and highly efficient bioluminescence, enabling rapid, non-invasive, and comprehensive assessment of biofilms together with quantification of a wide range of cell states. Although this method is limited to laboratory use due to the involvement of genetically modified organisms, it provides valuable insights into C. jejuni biofilm dynamics that could indirectly help in the development of improved food safety measures.

Human astroviruses pose a significant public health threat, especially to children, the elderly, and immunocompromised individuals. Nevertheless, these viruses remain largely understudied, with no approved antivirals or vaccines. This protocol focuses on leveraging reverse genetics (RG) and replicon systems to unravel the biology of MLB genotypes, a key group of neurotropic astroviruses. Using reverse genetics and replicon systems, we identified critical genetic deletions linked to viral attenuation and neurotropism, pushing forward vaccine development. We also uncovered novel replication mechanisms involving ER membrane interactions, opening doors to new antiviral targets. Reverse genetics and replicon systems are essential for advancing our understanding of astrovirus biology, identifying virulence factors, and developing effective treatments and vaccines to combat their growing public health impact.

Transfer RNAs (tRNAs), the essential adapter molecules in protein translation, undergo various post-transcriptional modifications. These modifications play critical roles in regulating tRNA folding, stability, and codon–anticodon interactions, depending on the modified position. Methods for detecting modified nucleosides in tRNAs include isotopic labeling combined with chromatography, antibody-based techniques, mass spectrometry, and high-throughput sequencing. Among these, high-performance liquid chromatography (HPLC) has been a cornerstone technique for analyzing modified nucleosides for decades. In this protocol, we provide a detailed, streamlined approach to purify and digest tRNAs from yeast cells and analyze the resulting nucleosides using HPLC. By assessing UV absorbance spectra and retention times, modified nucleosides can be reliably quantified with high accuracy. This method offers a simple, fast, and accessible alternative for studying tRNA modifications, especially when advanced technologies are unavailable.

Cricket paralysis virus (CrPV), a member of the family Dicistroviridae, is a single-stranded positive-sense RNA virus that primarily infects arthropods. Some members of the dicistrovirus family, including the honey bee viruses Israeli acute paralysis virus and Acute bee paralysis virus and the shrimp-infecting Taura syndrome virus, pose significant threats to agricultural ecosystems and economies worldwide. Dicistrovirus infection in Drosophila is used as a model system to study fundamental insect–virus–host interactions. The availability of a CrPV infectious clone allows controlled manipulation of the viral genome at a molecular level. Effective viral propagation and titration techniques are crucial for understanding the pathogenesis and epidemiology of dicistrovirus infections. Traditional methods for assessing viral titers, such as plaque assays, are unsuitable for CrPV, since Drosophila tissue culture cells like Schneider 2 cells cannot readily form adherent plaques. Here, we present a streamlined protocol for generating a recombinant virus from a CrPV infectious clone, propagating the virus in S2 cells and titering the virus by an immunofluorescence-based focus-forming assay (FFA). This protocol offers a rapid and reliable approach for generating recombinant viruses, viral amplification, and determining CrPV titers, enabling efficient investigation into viral biology and facilitating the development of antiviral strategies.

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.

Genome walking is a popular molecular technique for accessing unknown flanking DNAs, which has been widely used in biology-related fields. Herein, a simple but accurate genome-walking protocol named partially overlapping primer (POP)-based PCR (POP-PCR) is described. This protocol exploits a POP set of three POPs to mediate genome walking. The three POPs have a 10 nt 3' overlap and 15 nt heterologous 5' regions. Therefore, a POP can partially anneal to the previous POP site only at a relatively low temperature (approximately 50 °C). In primary POP-PCR, the low-temperature (25 °C) cycle allows the primary POP to partially anneal to site(s) of an unknown flank and many sites of the genome, synthesizing many single-stranded DNAs. In the subsequent high-temperature (65 °C) cycle, the target single-stranded DNA is converted into double-stranded DNA by the sequence-specific primer, attributed to the presence of this primer complement, while non-target single-stranded DNA cannot become double-stranded because it lacks a binding site for both primers. As a result, only the target DNA is amplified in the remaining 65 °C cycles. In secondary or tertiary POP-PCR, the 50 °C cycle directs the POP to the previous POP site and synthesizes many single-stranded DNAs. However, as in the primary PCR, only the target DNA can be amplified in the subsequent 65 °C cycles. This POP-PCR protocol has many potential applications, such as screening microbes, identifying transgenic sites, or mining new genetic resources.

Animal infection models play significant roles in the study of bacterial pathogenic mechanisms and host–pathogen interactions, as well as in evaluating drug and vaccine efficacies. Chlamydia trachomatis is responsible for infections in various mucosal tissues, including the eyes and urogenital, respiratory, and gastrointestinal tracts. Chronic infections can result in severe consequences such as trachoma-induced blindness, ectopic pregnancy, and infertility. While intravaginal inoculation of C. muridarum mimics the natural route of sexual transmission between individuals, transcervical inoculation allows the organisms to directly infect endometrial epithelial cells without interference from host responses triggered by chlamydial contact or infection of vaginal and cervical cells. Therefore, in this study, we used mouse models to visualize pathologies in both the endometrium and oviduct following C. muridarum inoculation.