Whole genome sequencing (WGS) of bacteria has become a routine method in diagnostic laboratories. One of the clinically most useful advantages of WGS is the ability to predict antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in bacterial sequences. This allows comprehensive investigations of such genetic features but can also be used for epidemiological studies. A plethora of software programs have been developed for the detailed annotation of bacterial DNA sequences, such as rapid annotation using subsystem technology (RAST), Resfinder, ISfinder, INTEGRALL and The Transposon Registry. Unfortunately, to this day, a reliable annotation tool of the combination of ARGs and MGEs is not available, and the generation of genbank files requires much manual input. Here, we present a new webserver which allows the annotation of ARGs, integrons and transposable elements at the same time. The pipeline generates genbank files automatically, which are compatible with Easyfig for comparative genomic analysis. Our BacAnt code and standalone software package are available at https://github.com/xthua/bacant with an accompanying web application at http://bacant.net.

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BacAnt: A Combination Annotation Server for Bacterial DNA Sequences to Identify Antibiotic Resistance Genes, Integrons, and Transposable Elements.

Metagenomics next-generation sequencing tests take the stage in the diagnosis of lower respiratory tract infections

Identifying the causes of community-acquired pneumonia (CAP) is challenging due to the disease’s complex etiology and the limitations of traditional microbiological diagnostic methods. Recent advances in next generation sequencing (NGS)-based metagenomics allow pan-pathogen detection in a single assay, and may have significant advantages over culture-based techniques. We conducted a cohort study of 159 CAP patients to assess the diagnostic performance of a clinical metagenomics assay and its impact on clinical management and patient outcomes. When compared to other techniques, clinical metagenomics detected more pathogens in more CAP cases, and identified a substantial number of polymicrobial infections. Moreover, metagenomics results led to changes in or confirmation of clinical management in 35 of 59 cases; these 35 cases also had significantly improved patient outcomes. Clinical metagenomics could be a valuable tool for the diagnosis and treatment of CAP. Trial registration number with the Chinese Clinical Trial Registry: ChiCTR2100043628
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Clinical metagenomics assessments improve diagnosis and outcomes in community-acquired pneumonia.

Infectious diseases are major threats to human health and lead to a serious public health burden. The emergence of new pathogens and the mutation of known pathogens challenge our ability to diagnose and control infectious diseases. Nanopore sequencing technology exhibited versatile applications in pathogenic microorganism detection due to its flexible data throughput. This review article introduced the applications of nanopore sequencing in clinical microbiology and infectious diseases management, including the monitoring of emerging infectious diseases outbreak, identification of pathogen drug resistance, and disease-related microbial communities characterization.

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The Applications of Nanopore Sequencing Technology in Pathogenic Microorganism Detection

Rapid and accurate etiologic diagnosis accelerates targeted antimicrobial therapy. Metagenomic analysis has played a critical role in pathogen identification. In this study, we leveraged the advantages of both the MinION and BGISEQ-500 platforms to make a bacteriologic diagnosis from a culture-negative lung tissue sample from an immunocompromised patient with severe pneumonia. Real-time nanopore sequencing rapidly identified Klebsiella pneumoniae by an 823 bp specific sequence within 1 min. Genomic analysis further identified bla SHV-12, bla KPC-2, bla TEM-1, bla CTX-M-65, and other resistance genes. The same sample was further sequenced on the BGISEQ-500 platform, which presented consistent results regarding the most top dominant pathogens and provided additional information of resistance genes. Revised antibiotic treatment was followed by the patient's clinical recovery. Though sample preparation and the interpretation of final results still need to be improved further, metagenomic sequencing contributes to the accurate diagnosis of culture-negative infections and facilitates the rational antibiotic therapy.

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Metagenomic Diagnosis for a Culture-Negative Sample From a Patient With Severe Pneumonia by Nanopore and Next-Generation Sequencing

Background: Enterobacter cloacae (E. cloacae) is a pathogen which is responsible for serious nosocomial infections. A gene which plays an important role in resistance to carbapenems in E. cloacae is new Delhi metallo-β-lactamase 1 (NDM-1). Currently, the spread of E. cloacae which carry a plasmid containing blaNDM-1 is a serious public threat. Methods: A multidrug-resistant clone of E. cloacae, CBG15936, was recovered in 2017 in Guangzhou, China. PCR, S1-pulsed-ﬁeld gel electrophoresis, and Southern blotting were performed to identify and locate the blaNDM-1 gene. Susceptibility testing and conjugation experiments were also performed. Illumina HiSeq and Nanopore sequencers were used to perform whole genome sequencing. Results: Strain CBG15936 belongs to ST932 and is resistant to carbapenems. Correspondingly, a blaNDM-1 gene was found in the IncN1 plasmid of this strain. This plasmid is approximately ~62 kb in length and has a conjugation frequency of 1.68 × 10−3 events per donor cell. A new transposon, Tn6696, was also identified on this plasmid. Tn6696 consists of an intact qnrS1-carrying Tn6292 element, an inverted 8.3-kb Tn3000 remnant, ISkpn19, ΔtnpA, and IS26. Conclusion: A new transposon, Tn6696, has been detected on a blaNDM-1-carrying plasmid recovered from multidrug-resistant E. cloacae CBG15936 from China. This finding provides a new perspective regarding the potential for blaNDM-1 to undergo horizontal transfer among drug-resistant bacteria.

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Qichao Chen

Papers

Metagenomic Diagnosis for a Culture-Negative Sample From a Patient With Severe Pneumonia by Nanopore and Next-Generation Sequencing

Characterization of a New Transposon, Tn6696, on a blaNDM-1-Carrying Plasmid From Multidrug-Resistant Enterobacter cloacae ssp. dissolvens in China.