Resolving plasmid-encoded carbapenem resistance dynamics and reservoirs in a hospital setting through nanopore sequencing

Carbapenem-resistance worldwide: a call for action - correspondence.

BackgroundChromosomal inversions are underappreciated causes of rare diseases given their detection, resolution, and clinical interpretation remain challenging. Heterozygous mutations in the MEIS2 gene cause an autosomal dominant syndrome characterized by intellectual disability, cleft palate, congenital heart defect, and facial dysmorphism at variable severity and penetrance.Case presentationHerein, we report a Chinese girl with intellectual disability, developmental delay, and congenital heart defect, in whom G-banded karyotype analysis identified a de novo paracentric inversion 46,XX, inv(15)(q15q26.1) and other conventional approaches including chromosomal microarray analysis and whole exome sequencing were failed to detect any pathologic variants that can explain the phenotypes of the proband. Subsequently, long-read Nanopore sequencing was directly conducted and defined the breakpoint position of the inversion, disrupting the MEIS2 gene at intron 8. These breakpoints were also confirmed by Sanger sequencing.ConclusionsIn conclusion, we report the first chromosomal inversion disrupting the MEIS2 gene, which was fine-mapped by long read Nanopore sequencing. Our data not only expand the clinical spectrum of MEIS2-caused syndromic developmental delay, but also illustrate the value of long-read sequencing in elucidating the precise genetic etiology of patients with relatively nonspecific clinical findings and chromosomal inversion that are beyond the resolution of conventional approaches.

U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E. coli in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.

Transmission of clones of carbapenem-resistant Escherichia coli between a hospital and an urban wastewater treatment plant

The rates of antibiotic resistance of Helicobacter pylori are increasing, and the patterns of resistance are region and population specific. Here, we elucidated the antibiotic resistance pattern of H. pylori in a single center in China and compared short-read- and long-read-based whole-genome sequencing for identifying the genotypes. Resistance rates of 38.5%, 61.5%, 27.9%, and 13.5% against clarithromycin, metronidazole, levofloxacin, and amoxicillin were determined, respectively, while no strain was resistant to tetracycline or furazolidone. Single nucleotide variations (SNVs) in the 23S rRNA and GyrA/B genes revealed by Illumina short-read sequencing showed good diagnostic abilities for clarithromycin and levofloxacin resistance, respectively. Nanopore long-read sequencing also showed a good efficiency in elucidating SNVs in the 23S rRNA gene and, thus, a good ability to detect clarithromycin resistance. The two technologies displayed good consistency in discovering SNVs and shared 76% of SNVs detected in the rRNA gene. Taking Sanger sequencing as the gold standard, Illumina short-read sequencing showed a slightly higher accuracy for discovering SNVs than Nanopore sequencing. There are two copies of the rRNA gene in the genome of H. pylori, and we found that the two copies were not the same in at least 26% of the strains tested, indicating their heterozygous status. Especially, three strains harboring a 2143G/A heterozygous status in the 23S rRNA gene, which is the most important site for clarithromycin resistance, were found. In conclusion, our results provide evidence for an empirical first-line treatment for H. pylori eradication in clinical settings. Moreover, we show that Nanopore sequencing is a potential tool for predicting clarithromycin resistance. IMPORTANCE Helicobacter pylori resistance has been increasing in recent years. The resistance profile, which is important for empirical treatment, is region and population specific. We found high rates of resistance to metronidazole, clarithromycin, and levofloxacin in H. pylori in our center, while no resistance to tetracycline or furazolidone was found. These results provide a reference for local physicians prescribing antibiotics for H. pylori eradication. Nanopore sequencing recently appeared to be a promising technology for elucidating whole-genome sequences, which generates long sequencing reads and is time-efficient and portable. However, a relatively higher error rate of sequencing reads was also found. In this study, we compared Nanopore sequencing and Illumina sequencing for revealing single nucleotide variations in the 23S rRNA gene, which determines clarithromycin resistance, and we found that although there were a few false discoveries, Nanopore sequencing showed good consistency with Illumina sequencing, indicating that it is a potential tool for predicting clarithromycin resistance.

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

Background & objectives:The growing incidence and the wide diversity of carbapenemase-producing bacterial strains is a major concern as only a few antimicrobial agents are active on carbapenem-resistant bacteria. This study was designed to study molecular epidemiology of carbapenem-resistant Gram-negative bacterial (GNB) isolates from the community and hospital settings.Methods:In this study, non-duplicate GNB were isolated from clinical specimens, and phenotypic test such as modified Hodge test, metallo β-lactamase E-strip test, etc. were performed on carbapenem-resistant bacteria. Multiplex PCR was performed to identify the presence of blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM. Minimum inhibitory concentration (MIC) of colistin, fosfomycin, minocycline, chloramphenicol and tigecycline was also determined.Results:Of the 3414 GNB studied, carbapenem resistance was 9.20 per cent and maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4%) and primary centre (2.1%). Among the carbapenem-resistant bacteria, overall, the most common isolate was Pseudomonas aeruginosa (24%). On multiplex PCR 90.3 per cent carbapenem-resistant isolates were positive for carbapenemase gene. The blaNDM (63%) was the most prevalent gene followed by blaVIM (18.4%). MIC results showed that 88 per cent carbapenem-resistant Enterobacteriaceae were sensitive to fosfomycin, whereas 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin.Interpretation & conclusions:Carbapenem resistance in GNB isolates from the community and hospital settings was found to be on the rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, prudent use of antibiotics and strict infection control practices should be followed in hospital to limit the emergence and spread of multidrug-resistant bacteria.

The coronavirus (COVID-19) global pandemic has impacted the health of almost everyone, including changes in their salivary microbiota. Since 2019, there has been an increase in the number of new COVID-19 cases in Thailand. Therefore, COVID-19 active case finding is important for early detection and epidemic control. Moreover, the dynamic changes of salivary bacteriome in asymptomatic COVID-19 cases are largely unknown. This research aimed to investigate and compare the salivary bacteriome and the co-infectious bacterial pathogens in the asymptomatic COVID-19 positive group to the negative group, based on novel nanopore sequencing. This cohort was a cross-sectional study including saliva samples collected from 82 asymptomatic participants (39 COVID-19 positive and 43 COVID-19 negative cases). All samples were sequenced for the full-length bacterial 16S rDNA. The alpha and beta diversity analyses were not significantly different between groups. The three major species in salivary bacteriome including Veillonella parvula, Streptococcus mitis, and Prevotella melaninogenica were observed in both groups. Interestingly, Lautropia mirabilis was a significantly enriched species in the saliva of the asymptomatic COVID-19-positive cases based on linear discriminant analysis effect size (LEfSe) analysis. The results suggested that L. mirabilis was a co-infectious agent in the asymptomatic COVID-19 group. However, the potential role of L. mirabilis should be validated in further experimental studies.

We present the complete closed circular genome sequence derived from Oxford Nanopore sequencing of the shipworm endosymbiont Teredinibacter turnerae T7902 (DSM 15152, ATCC 39867), originally isolated from the shipworm Lyrodus pedicellatus (1). This sequence will aid in the comparative genomics of shipworm endosymbionts and the understanding of host-symbiont evolution.

We present the complete closed circular genome sequence derived from the Oxford Nanopore sequencing of the shipworm endosymbiont, Teredinibacter turnerae T7902 (DSM 15152, ATCC 39867), originally isolated from the shipworm, Lyrodus pedicellatus (1). This sequence will aid in the comparative genomics of shipworm endosymbionts and the understanding of the host-symbiont evolution.

Our aim was to analyze mortality attributable to carbapenem-resistant (CR) gram-negative bacilli (GNB) in patients with bloodstream infections (BSIs). Prospective multicentric study including patients with GNB-BSI from 19 Italian hospitals (June 2018-January 2020). Patients were followed-up to 30 days. Primary outcomes were 30-day mortality and attributable mortality. Attributable mortality was calculated in the following groups: Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales, metallo-β-lactamases (MBL)-producing Enterobacterales, CR-Pseudomonas aeruginosa (CRPA), CR-Acinetobacter baumannii (CRAB). A multivariable analysis with hospital fixed-effect was built to identify factors associated with 30-day mortality. Adjusted OR (aORs) were reported. Attributable mortality was calculated according to the DRIVE-AB Consortium. Overall, 1276 patients with monomicrobial GNB BSI were included: 723/1276 (56.7%) carbapenem-susceptible (CS)-GNB, 304/1276 (23.8%) KPC-, 77/1276 (6%) MBL-producing CRE, 61/1276 (4.8%) CRPA, and 111/1276 (8.7%) CRAB BSI. Thirty-day mortality in patients with CS-GNB BSI was 13.7% compared to 26.6%, 36.4%, 32.8% and 43.2% in patients with BSI by KPC-CRE, MBL-CRE, CRPA and CRAB, respectively (P < .001). On multivariable analysis, age, ward of hospitalization, SOFA score, and Charlson Index were factors associated with 30-day mortality, while urinary source of infection and early appropriate therapy resulted protective factors. Compared to CS-GNB, MBL-producing CRE (aOR 5.86, 95% CI 2.72-12.76), CRPA (aOR 1.99, 95% CI 1.48-5.95) and CRAB (aOR 2.65, 95% CI 1.52-4.61) were significantly associated with 30-day mortality. Attributable mortality rates were 5% for KPC-, 35% for MBL, 19% for CRPA, and 16% for CRAB. In patients with BSIs, carbapenem-resistance is associated with an excess of mortality, with MBL-producing CRE carrying the highest risk of death.

BackgroundIn various neurodevelopmental disorders (NDDs), sets of differential methylation marks (referred to as DNA methylation signatures or episignatures) are syndrome-specific and useful in evaluating the pathogenicity of detected genetic variants. These signatures have generally been tested using methylation arrays, requiring additional experimental and evaluation costs. As an alternative, long-read sequencing can simultaneously and accurately evaluate genetic and epigenetic changes. In addition, genome-wide DNA methylation profiling with more complete sets of CpG using long-read sequencing (than methylation arrays) may provide alternative but more comprehensive DNA methylation signatures, which have yet to be adequately investigated.MethodsNine and seven cases of molecularly diagnosed Sotos syndrome and ATR-X syndrome, respectively, were sequenced using nanopore long-read sequencing, together with 22 controls. Genome-wide differential DNA methylation analysis was performed. Among these differential DNA methylation sites, a single-locus DNA methylation mark at part of the NSD1 CpG island (CpGi) was subsequently studied in an additional 22 cases with a NSD1 point mutation or a 5q35 submicroscopic deletion involving NSD1. To investigate the potential utility of a single-locus DNA methylation test at NSD1 CpGi for differential diagnosis, nine cases with NSD1-negative clinically overlapping overgrowth intellectual disability syndromes (OGIDs) were also tested.ResultsLong-read sequencing enabled the successful extraction of two sets of differential methylation marks unique to each of Sotos syndrome and ATR-X syndrome, referred to as long-read-based DNA methylation signatures (LR-DNAm signatures), as alternatives to reported DNA methylation signatures (obtained by methylation array). Additionally, we found that a part, but not all, of the NSD1 CpGi were hypomethylated compared with the level in controls in both cases harboring NSD1 point mutations and those with a 5q35 submicroscopic deletion. This difference in methylation is specific to Sotos syndrome and lacking in other OGIDs.ConclusionsSimultaneous evaluation of genetic and epigenetic alterations using long-read sequencing may improve the discovery of DNA methylation signatures, which may in turn increase the diagnostic yields. As an example of the outcomes of these analyses, we propose that a single-locus DNA methylation test at NSD1 CpGi may streamline the molecular diagnosis of Sotos syndrome, regardless of the type of NSD1 aberration.

Genomic investigation of NDM-1 producing Enterobacterales transmission in a South Korean hospital.

died at 5 dpi. The dead piglets showed similar lesions to those of the piglets infected with YS isolate in the stomach, intestines, and mesenteric lymph nodes. Piglets in the control group orally inoculated with Dulbecco minimal essential medium remained healthy during the experiment, and no obvious pathologic changes were observed.

Carbapenem-resistant Enterobacteriaceae (CRE) have become a public health threat worldwide. There are three major mechanisms by which Enterobacteriaceae become resistant to carbapenems: enzyme production, efflux pumps and porin mutations. Of these, enzyme production is the main resistance mechanism. There are three main groups of enzymes responsible for most of the carbapenem resistance: KPC (Klebsiella pneumoniae carbapenemase) (Ambler class A), MBLs (Metallo-ß-Lactamases) (Ambler class B) and OXA-48-like (Ambler class D). KPC-producing Enterobacteriaceae are endemic in the United States, Colombia, Argentina, Greece and Italy. On the other hand, the MBL NDM-1 is the main carbapenemase-producing resistance in India, Pakistan and Sri Lanka, while OXA-48-like enzyme-producers are endemic in Turkey, Malta, the Middle-East and North Africa. All three groups of enzymes are plasmid-mediated, which implies an easier horizontal transfer and, thus, faster spread of carbapenem resistance worldwide. As a result, there is an urgent need to develop new therapeutic guidelines to treat CRE infections. Bearing in mind the different mechanisms by which Enterobacteriaceae can become resistant to carbapenems, there are different approaches to treat infections caused by these bacteria, which include the repurposing of already existing antibiotics, dual therapies with these antibiotics, and the development of new ß-lactamase inhibitors and antibiotics.