The emergence of carbapenemase-producing Citrobacter freundii poses a significant threat to public health worldwide. Here, we reported a C. freundii strain CWH001 which was resistant to all tested antimicrobials except tetracycline. Whole genome sequencing and analysis were performed. The strain, which belonged to a new sequence type ST139, showed close relationship with other foreign C. freundii strains through phylogenetic analysis. A novel variant of the intrinsic bla_CMY gene located on the chromosome was identified and designated as bla_CMY-152. Coexistence of bla_NDM-1 with qnrS1 was found on a conjugative IncN plasmid, which had a backbone appearing in various plasmids. Other class A ESBL genes (bla_VEB-3 and bla_TEM-1) were also detected on two different novel plasmids. The emergence of multidrug-resistant C. freundii is of major concern, causing great challenges to the treatment of clinical infections. Great efforts need to be taken for the specific surveillance of this opportunistic pathogen.

We have recovered one blaNDM-1-harboring bacterial strain, designated as XM1570, from a sputum sample obtained from a fatal case of pneumonia in China. Biochemical profiling, 16S rRNA sequencing and antimicrobial susceptibility testing were performed. Conjugation experiments were conducted to determine transmissibility of resistance. Pulsed-field gel electrophoresis and whole genome sequencing were performed to identify strain-specific features. The isolate XM1570 was identified as Acinetobacter calcoaceticus. Whole genome sequencing identified two plasmids, pXM1 and pXM2. Comparative analysis showed >99% similarity between XM1570 and A. calcoaceticus PHEA-2. Plasmid pXM1 carried the carbapenemase gene blaNDM-1 and displayed high homology with previously described plasmids isolated from different Acinetobacter spp., which were collected from human or livestock distributed in China and worldwide. The blaNDM-1 gene was located on this conjugative plasmid in a transposon-like region flanked by two copies of the insertion sequence ISAba125; and resistance to all tested β-lactams was observed. Transferability of resistance from pXM1 to the transconjugants was identified. Plasmid pXM2 had an insertion sequence ISAba125 and a −35 region of the blaNDM-1 gene promoter but the blaNDM-1 gene was not present. A chromosomally located carbapenemase-encoding gene blaOXA-75 was detected; however, this gene was interrupted by an insertion sequence ISAba22 belonging to IS3 family. Location of blaNDM-1 on different self-transmissible plasmids could facilitate geographically broad dissemination and host range expansion of the blaNDM-1 gene via horizontal gene transfer. Our findings of this normally environmental species A. calcoaceticus XM1570 further underline the significant clinical challenge and the essential need for surveillance including molecular methods and plasmid analyses.

Nanopore sequencing has been widely used for the real-time detection and surveillance of pathogens with portable MinION. Nanopore adaptive sequencing can enrich on-target sequences without additional pretreatment. In this study, the performance of adaptive sequencing was evaluated for viral genome enrichment of clinical respiratory samples. Ligation-based nanopore adaptive sequencing (LNAS) and rapid PCR-based nanopore adaptive sequencing (RPNAS) workflows were performed to assess the effects of enrichment on nasopharyngeal swab samples from human adenovirus (HAdV) outbreaks. RPNAS was further applied for the enrichment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal swab samples to evaluate sensitivity and timeliness. The RPNAS increased both the relative abundance (7.87–12.86-fold) and data yield (1.27–2.15-fold) of HAdV samples, whereas the LNAS increased only the relative abundance but had no obvious enrichment on the data yield. Compared with standard nanopore sequencing, RPNAS detected the SARS-CoV-2 reads from two low-abundance samples, increased the coverage of SARS-CoV-2 by 36.68–98.92%, and reduced the time to achieve the same coverage. Our study highlights the utility of RPNAS for virus enrichment directly from clinical samples, with more on-target data and a shorter sequencing time to recover viral genomes. These findings promise to improve the sensitivity and timeliness of rapid identification and genomic surveillance of infectious diseases.

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.

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) plays an important role in COVID‐19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS‐CoV‐2 followed by either massively parallel or nanopore sequencing is a widely‐used strategy to obtain genome sequences from primary samples. However, multiplex PCR‐based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two‐pool, long‐amplicon 36‐plex PCR primer panel with ~1000‐bp amplicon lengths for full‐genome sequencing of SARS‐CoV‐2. We validated the panel by assessing nasopharyngeal swab samples with a 95% viral genome coverage with a ≥10‐fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false‐positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5′ or 3′ ends of reads. Thus, nanopore sequencing could not elucidate intra‐host viral diversity.

Background: The New Delhi metallo-β-lactamase-1 (NDM-1)-positive plasmid and its variants pose daunting threats to public health. Hospital sewage was considered as an important reservoir of antibiotic genes. Numerous and diverse taxa of multidrug-resistant (MDR) bacteria carrying NDM-1-positive plasmids have been identified during routine surveillance of hospital sewage. We herein report a carbapenem-resistant Acinetobacter towneri strain AeBJ009 with an NDM-1-positive plasmid isolated from hospital sewage. Materials and Methods: Bacteria were isolated from cultures of hospital sewage and identified by using the Vitek 2 compact system and 16S rRNA sequencing. The bla_NDM-1 gene was amplified and confirmed by sequencing. Antimicrobial susceptibility testing was performed using AST-GN14 on the Vitek2 compact system. In addition, the bla_NDM-1 gene was located by Southern blotting. Conjugation experiment and whole-genome sequencing were performed for further analysis. Results: Strain AeBJ009 was isolated from hospital sewage and identified as A. towneri. Antimicrobial susceptibility testing revealed an MDR phenotype. Pulsed-field gel electrophoresis and Southern blotting showed that strain AeBJ009 carries three plasmids and that bla_NDM-1 is located on the 47kb plasmid pNDM-AeBJ009. However, the conjugation experiment to transfer pNDM-AeBJ009 to Escherichia coli strain J53 was unsuccessful. Whole-genome sequencing found that pNDM-AeBJ009 contains a Tn 125 element carrying bla_NDM-1. The ble gene downstream of bla_NDM-1 displayed a single-nucleotide polymorphism compared to its homologue on plasmid pM131_NDM1. BLAST analysis using the Comprehensive Antibiotic Resistance Database identified no gene polymorphisms with 100% identity to our ble variant. Conclusion: The A. towneri strain AeBJ009 exhibiting an extended spectrum of antibiotic resistance was isolated from hospital sewage and may potentially exacerbate the risk of MDR bacterial infections. The prevention of nosocomial infections due to drug-resistant bacteria will require enhanced monitoring and control of MDR pathogens in environmental reservoirs.