Pre-isolation Molecular Screening for High-throughput Sampling and Sequencing of Bacterial Microbes from the Environment.

Environmental studies often require culture and characterization to understand the prevalence, distribution, persistence and functions of target microorganisms in ecological habitats. Isolating pure microbiological monocultures allows the phenotypic characterization of microorganisms to study their functional properties. For efficient isolation of low-prevalence organisms, enrichment followed by PCR screening is performed to identify positive samples for subsequent culture. Molecular characterization, strain-typing, and genotyping of isolated microorganisms is best comprehensively performed using whole-genome sequencing. This article outlines end-to-end protocols for screening, isolation, and sequencing of microbes from environmental samples. We provide systematic methods for environmental study design, enrichment, screening, and isolation of target microorganisms. Species identification is performed using qPCR or MALDI-TOF MS. Genomic DNA is extracted for whole-genome sequencing using the Oxford Nanopore platform. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Designing and conducting an environmental soil sampling study Basic Protocol 2: Enrichment of microbes from environmental soil samples Alternate Protocol 1: Collection and enrichment of microbes from environmental water samples Basic Protocol 3: Screening of enriched samples by direct qPCR Basic Protocol 4: Enumeration and isolation of enriched samples using selective medium Basic Protocol 5: Species confirmation using colony qPCR Alternate Protocol 2: Species identification using a MALDI-TOF MS Biotyper Alternate Protocol 3: Species identification of bacterial isolates using universal PCR primers and Sanger sequencing Basic Protocol 6: Cryostorage of bacterial isolates Basic Protocol 7: Extraction of genomic DNA Basic Protocol 8: Quality check of extracted genomic DNA Basic Protocol 9: Whole-genome sequencing using the Oxford Nanopore MinION Platform.

Draft Genome Sequence of Multidrug Resistant Klebsiella pneumoniae Strain C43 Isolated from Environmental Water Sample.

Here, we report the genome of ESBL-producing Klebsiella pneumoniae strain C43, which was isolated from an environmental water sample. The genome is 5,614,412 bp in size with GC content of 56.86% with multidrug antimicrobial resistance genes and several metal resistance gene operons.

GALAXY Workflow for Bacterial Next-Generation Sequencing De Novo Assembly and Annotation.

Whole-genome sequencing of prokaryotes is now readily available and affordable on next-generation sequencing platforms. However, the process of de novo assembly can be complicated and tedious for those without a background in computational biology, bioinformatics, or UNIX. Licenses for commercial bioinformatics software may be costly and limited in flexibility. GALAXY is a powerful graphical open-source code-free bioinformatics platform that is freely available on multiple public and private servers. Here, we describe a bacterial de novo assembly workflow using GALAXY. It performs de novo genome assembly using short reads, long reads, or a hybrid method using both short and long reads. Genome annotation, prediction of antimicrobial resistance genes, and multi-locus sequence typing are subsequently performed to characterize the draft genome. Performing genome assembly and annotation on this pipeline allows documentation, parameterization, and sharing, facilitating replication, reuse, and reproducibility of both data and methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Quality check of NGS reads Basic Protocol 2: De novo assembly using Unicycler Basic Protocol 3: Assembly quality check using QUAST and Bandage Basic Protocol 4: Genome annotation using Prokka Basic Protocol 5: Prediction of antimicrobial resistance genes (ARGs) Basic Protocol 6: Multi-locus sequence typing (MLST).

Draft Genome Sequence of Enterobacter hormaechei subsp. steigerwaltii Strain BEI01.

Here, we report the genome sequence of Enterobacter hormaechei subsp. steigerwaltii strain BEI01, originally deposited as a member of the Enterobacter cloacae complex. The genome is 4,900,246 bp in size with a GC content of 55.44%; it contains multidrug antimicrobial resistance genes and several metal resistance gene operons.

Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler.

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.