scholarly journals Nanopore sequencing for fast determination of plasmids, phages, virulence markers, and antimicrobial resistance genes in Shiga toxin-producingEscherichia coli

2019 ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Marc A. Allard ◽  
Eric W. Brown ◽  
Shashi Sharma ◽  
Maria Hoffmann
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Single Chromosome ◽  
Short Read ◽  
Virulence Markers ◽  
Antimicrobial Resistance Genes ◽  
Public Health Information ◽  
Long Read ◽  
Positive Correlations ◽  
Rapid Characterization

AbstractWhole genome sequencing can provide essential public health information. However, it is now known that widely used short-read methods have the potential to miss some randomly-distributed segments of genomes. This can prevent phages, plasmids, and virulence factors from being detected or properly identified. Here, we compared assemblies of three complete STEC O26:H11 genomes from two different sequence types (ST21 and 29), each acquired using the MiSeq-Nextera XT, MinION nanopore-based sequencing, and Pacific Biosciences (PacBio) sequencing. Each closed genome consisted of a single chromosome, approximately 5.7 Mb for CFSAN027343, 5.6 Mb for CFSAN027346, and 5.4 MB for CFSAN027350. However, short-read WGS using MiSeq-Nextera failed to identify some virulence genes in plasmids and on the chromosome, both of which were detected using the long-read platforms. Results from long-read MinION and PacBio allowed us to identify differences in plasmid content: a single 88 kb plasmid in CFSAN027343; a 157kb plasmid in CFSAN027350; and two plasmids in CFSAN027346 (one 95 Kb, one 72 Kb). These data enabled rapid characterization of the virulome, detection of antimicrobial genes, and composition/location of Stx phages. Taken together, positive correlations between the two long-read methods for determining plasmids, virulome, antimicrobial resistance genes, and phage composition support MinION sequencing as one accurate and economical option for closing STEC genomes and identifying specific virulence markers.

scholarly journals Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation

2018 ◽  
Author(s):  
Derek M. Bickhart ◽  
Mick Watson ◽  
Sergey Koren ◽  
Kevin Panke-Buisse ◽  
Laura M. Cersosimo ◽  
...  
Keyword(s):  
Microbial Community ◽  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Open Reading Frames ◽  
Short Read ◽  
Proximity Ligation ◽  
Antimicrobial Resistance Genes ◽  
Long Read ◽  
Assembly Algorithms

AbstractThe characterization of microbial communities by metagenomic approaches has been enhanced by recent improvements in short-read sequencing efficiency and assembly algorithms. We describe the results of adding long-read sequencing to the mix of technologies used to assemble a highly complex cattle rumen microbial community, and compare the assembly to current short read-based methods applied to the same sample. Contigs in the long-read assembly were 7-fold longer on average, and contained 7-fold more complete open reading frames (ORF), than the short read assembly, despite having three-fold lower sequence depth. The linkages between long-read contigs, provided by proximity ligation data, supported identification of 188 novel viral-host associations in the rumen microbial community that suggest cross-species infectivity of specific viral strains. The improved contiguity of the long-read assembly also identified 94 antimicrobial resistance genes, compared to only seven alleles identified in the short-read assembly. Overall, we demonstrate a combination of experimental and computational methods that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

scholarly journals Differential overlap in human and animal fecal microbiomes and resistomes in rural versus urban Bangladesh

2021 ◽  
Author(s):  
Jenna M Swarthout ◽  
Erica R Fuhrmeister ◽  
Latifah Hamzah ◽  
Angela Harris ◽  
Mir A. Ahmed ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Urban Communities ◽  
Resistance Genes ◽  
Low Income ◽  
Urban Areas ◽  
Pathogenic Bacteria ◽  
Small Scale ◽  
Rrna Gene ◽  
Antimicrobial Resistance Genes ◽  
Long Read

Background Low- and middle-income countries (LMICs) bear the largest mortality burden due to antimicrobial-resistant infections. Small-scale animal production and free-roaming domestic animals are common in many LMICs, yet data on zoonotic exchange of gut bacteria and antimicrobial resistance genes (ARGs) in low-income communities are sparse. Differences between rural and urban communities in population density, antibiotic use, and cohabitation with animals likely influence the frequency of transmission of gut bacterial communities and ARGs between humans and animals. Here, we determined the similarity in gut microbiomes, using 16S rRNA gene amplicon sequencing, and resistomes, using long-read metagenomics, between humans, chickens, and goats in rural compared to urban Bangladesh. Results Gut microbiomes were more similar between humans and chickens in rural (where cohabitation is more common) compared to urban areas, but there was no difference for humans and goats. Urbanicity did not impact the similarity of human and animal resistomes; however, ARG abundance was higher in urban animals compared to rural animals. We identified substantial overlap of ARG alleles in humans and animals in both settings. Humans and chickens had more overlapping ARG alleles than humans and goats. All fecal hosts carried ARGs on contigs classified as potentially pathogenic bacteria, including Escherichia coli, Campylobacter jejuni, Clostridiodes difficile, and Klebsiella pneumoniae. Conclusions While the development of antimicrobial resistance in animal gut microbiomes and subsequent transmission to humans has been demonstrated in intensive farming environments and high-income countries, evidence of zoonotic exchange of antimicrobial resistance in LMIC communities is lacking. This research provides genomic evidence of overlap of antimicrobial resistance genes between humans and animals, especially in urban communities, and highlights chickens as important reservoirs of antimicrobial resistance. Chicken and human gut microbiomes were more similar in rural Bangladesh, where cohabitation is more common. Incorporation of long-read metagenomics enabled characterization of bacterial hosts of resistance genes, which has not been possible in previous culture-independent studies using only short-read sequencing. These findings highlight the importance of developing strategies for combatting antimicrobial resistance that account for chickens being reservoirs of ARGs in community environments, especially in urban areas.

scholarly journals Non-human primates in the Gambia harbour human-associated pathogenic Escherichia coli strains

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Ebenezer Foster-Nyarko ◽  
Nabil-Fareed Alikhan ◽  
Anuradha Ravi ◽  
Gaëtan Thilliez ◽  
Nicholas Thomson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
The Gambia ◽  
Public Health Importance ◽  
Software Environment ◽  
E Coli ◽  
Antimicrobial Resistance Genes ◽  
Long Read ◽  
Sequence Types

Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between different host species. We have investigated genetic diversity and antimicrobial resistance in Escherichia coli isolates from a range of non-human primates dispersed across the Gambia: patas monkey (n=1), western colobus monkey (n=6), green monkey (n=14) and guinea baboon (n=22). From 43 stools, we recovered 99 isolates. We performed Illumina whole-genome shotgun sequencing on all isolates and nanopore long-read sequencing on isolates with antimicrobial resistance genes. We inferred the evolution of E. coli in this population using the EnteroBase software environment. We identified 43 sequence types (ten of them novel), spanning five of the eight known phylogroups of E. coli. Many of the observed sequence types and phylotypes from non-human primates have been associated with human extra-intestinal infection and carry virulence characteristics associated with disease in humans, particularly ST73, ST217 and ST681. However, we found a low prevalence of antimicrobial resistance genes in isolates from non-human primates. Hierarchical clustering showed that ST442 and ST349 from non-human primates are closely related to isolates from human infections, suggesting recent exchange of bacteria between humans and monkeys. Our results are of public health importance, considering the increasing contact between humans and wild primates.

scholarly journals Complete genome assembly of clinical multidrug resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial resistance genes and plasmids

2019 ◽  
Author(s):  
Thomas V. Sydenham ◽  
Søren Overballe-Petersen ◽  
Henrik Hasman ◽  
Hannah Wexler ◽  
Michael Kemp ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Complete Genome ◽  
Multidrug Resistant ◽  
Whole Genome ◽  
Short Read ◽  
Is Elements ◽  
Oxford Nanopore ◽  
Antimicrobial Resistance Genes ◽  
Genome Assemblies

ABSTRACTBacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance and the prevalence of antimicrobial resistance genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of antimicrobial resistance. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of antimicrobial resistance and comprehensive sequence databases support microbiome and metagenomic studies. Here we test several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu corrected ONT reads produced the assembly of highest quality. This approach was then applied to six clinical multidrug resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of antimicrobial resistance genes, however there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing antimicrobial resistance prediction for B. fragilis from whole genome sequencing.REPOSITORIESSequence files (MinION reads de-multiplexed with Deepbinner and basecalled with Albacore in fast5 format and Illumina MiSeq reads in fastq format) and final genome assemblies have been deposited to NCBI/ENA/DDBJ under Bioproject accessions PRJNA525024, PRJNA244942, PRJNA244943, PRJNA244944, PRJNA253771, PRJNA254401, and PRJNA254455IMPACT STATEMENTBacterial whole genome sequencing is increasingly used in public health, clinical, and research laboratories for typing, identification of virulence factors, phylogenomics, outbreak investigation and identification of antimicrobial resistance genes. In some settings, diagnostic microbiome amplicon sequencing or metagenomic sequencing directly from clinical samples is already implemented and informs treatment decisions. The prospect of prediction of antimicrobial susceptibility based on resistome identification holds promises for shortening time from sample to report and informing treatment decisions. Databases with comprehensive reference sequences of high quality are a necessity for these purposes. Bacteroides fragilis is an important part of the human commensal gut microbiota and is also the most commonly isolated anaerobic bacterium from non-faecal clinical samples but few complete genome assemblies are available through public databases. The fragmented assemblies from short read de novo assembly often negate the identification of insertion sequences upstream of antimicrobial resistance gens, which is necessary for prediction of antimicrobial resistance from whole genome sequencing. Here we test multiple assembly pipelines with short read Illumina data and long read data from Oxford Nanopore Technologies MinION sequencing to select an optimal pipeline for complete genome assembly of B. fragilis. However, B. fragilis is a highly plastic genome with multiple inversive repeat regions, and complete genome assembly of six clinical multidrug resistant isolates still required minor manual finishing for half the isolates. Complete identification of known insertion sequences and resistance genes was possible from the complete genome. In addition, the current catalogue of Bacteroides plasmid sequences is augmented by eight new plasmid sequences that do not have corresponding, complete entries in the NCBI database. This work almost doubles the number of publicly available complete, finished chromosomal and plasmid B. fragilis sequences paving the way for further studies on antimicrobial resistance prediction and increased quality of microbiome and metagenomic studies.DATA SUMMARYSequence read files (Oxford Nanopore (ONT) fast5 files and Illumina fastq files) as well as the final genome assemblies have been deposited to NCBI/ENA/DDBJ under Bioproject accessions PRJNA525024, PRJNA244942, PRJNA244943, PRJNA244944, PRJNA253771, PRJNA254401, and PRJNA254455.Fastq format of demultiplexed ONT reads trimmed of adapters and barcode sequences are available at doi.org/10.5281/zenodo.2677927Genome assemblies from the assembly pipeline validation are available at doi: doi.org/10.5281/zenodo.2648546.Genome assemblies corresponding to each stage of the process of the assembly are available at doi.org/10.5281/zenodo.2661704.Full commands and scripts used are available from GitHub: https://github.com/thsyd/bfassembly as well as a static version at doi.org/10.5281/zenodo.2683511

scholarly journals Long-Read Sequencing Reveals Evolution and Acquisition of Antimicrobial Resistance and Virulence Genes in Salmonella enterica

2021 ◽  
Vol 12 ◽  
Author(s):  
Cong Li ◽  
Gregory H. Tyson ◽  
Chih-Hao Hsu ◽  
Lucas Harrison ◽  
Errol Strain ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Salmonella Enterica ◽  
Heavy Metal Resistance ◽  
Multidrug Resistant ◽  
Metal Resistance ◽  
Virulence Plasmids ◽  
Antimicrobial Resistance Genes ◽  
Long Read ◽  
High Level

Salmonella enterica is a significant and phylogenetically diverse zoonotic pathogen. To understand its genomic heterogeneity and antimicrobial resistance, we performed long-read sequencing on Salmonella isolated from retail meats and food animals. A collection of 134 multidrug-resistant isolates belonging to 33 serotypes were subjected to PacBio sequencing. One major locus of diversity among these isolates was the presence and orientation of Salmonella pathogenic islands (SPI), which varied across different serotypes but were largely conserved within individual serotypes. We also identified insertion of an IncQ resistance plasmid into the chromosome of fourteen strains of serotype I 4,[5],12:i:– and the Salmonella genomic island 1 (SGI-1) in five serotypes. The presence of various SPIs, SGI-1 and integrated plasmids contributed significantly to the genomic variability and resulted in chromosomal resistance in 55.2% (74/134) of the study isolates. A total of 93.3% (125/134) of isolates carried at least one plasmid, with isolates carrying up to seven plasmids. We closed 233 plasmid sequences of thirteen replicon types, along with twelve hybrid plasmids. Some associations between Salmonella isolate source, serotype, and plasmid type were seen. For instance, IncX plasmids were more common in serotype Kentucky from retail chicken. Plasmids IncC and IncHI had on average more than five antimicrobial resistance genes, whereas in IncX, it was less than one per plasmid. Overall, 60% of multidrug resistance (MDR) strains that carried >3 AMR genes also carried >3 heavy metal resistance genes, raising the possibility of co-selection of antimicrobial resistance in the presence of heavy metals. We also found nine isolates representing four serotypes that carried virulence plasmids with the spv operon. Together, these data demonstrate the power of long-read sequencing to reveal genomic arrangements and integrated plasmids with a high level of resolution for tracking and comparing resistant strains from different sources. Additionally, the findings from this study will help expand the reference set of closed Salmonella genomes that can be used to improve genome assembly from short-read data commonly used in One Health antimicrobial resistance surveillance.

scholarly journals Detection of plasmid contigs in draft genome assemblies using customized Kraken databases

2020 ◽  
Author(s):  
Ryota Gomi ◽  
Kelly L. Wyres ◽  
Kathryn E. Holt
Keyword(s):  
Antimicrobial Resistance ◽  
Sensitivity And Specificity ◽  
Resistance Genes ◽  
Large Scale ◽  
Simple Procedure ◽  
Draft Genome ◽  
Short Read ◽  
Contig Length ◽  
Antimicrobial Resistance Genes ◽  
Genome Assemblies

ABSTRACTPlasmids play an important role in bacterial evolution and mediate horizontal transfer of genes including virulence and antimicrobial resistance genes. Although short-read sequencing technologies have enabled large-scale bacterial genomics, the resulting draft genome assemblies are often fragmented into hundreds of discrete contigs, which makes detailed characterization of plasmids difficult. Several tools and approaches have been developed to identify plasmid sequences in such assemblies, but require trade-off between sensitivity and specificity. Here we propose using the Kraken classifier, together with a custom Kraken database comprising known chromosomal and plasmid sequences of Klebsiella pneumoniae species complex (KpSC), to identify plasmid-derived contigs in draft assemblies. We assessed performance using Illumina-based draft genome assemblies for 82 KpSC isolates, for which complete genomes were available to supply ground truth. When benchmarked against five other classifiers (Centrifuge, RFPlasmid, mlplasmids, PlaScope, and Platon), Kraken showed balanced performance in terms of overall sensitivity and specificity (90.8% and 99.4%, respectively for contig count; 96.5% and >99.9%, respectively for cumulative contig length), and the highest accuracy (96.8% vs 91.8%-96.6% for contig count; 99.8% vs 99.0%-99.7% for cumulative contig length), and F1 score (94.5% vs 84.5%-94.1%, for contig count; 98.0% vs 88.9%-96.7% for cumulative contig length). Kraken was also among the most consistent performers at the individual genome level. Furthermore, we demonstrate that expanding the Kraken database with additional known chromosomal and plasmid sequences (a simple procedure that unlike other methods does not require any model training) can further improve classification performance. Although we have focused here on the KpSC, this methodology could easily be applied to other species with a sufficient number of completed genomes.IMPACT STATEMENTThe assembly of bacterial genomes using short-read data often results in hundreds of discrete contigs due to the presence of repeat sequences in those genomes. Separating plasmid contigs from chromosomal contigs in such assemblies is required, e.g., to assess the mobility of antimicrobial resistance genes. Although several tools have been developed for that purpose, they often suffer from low sensitivity or specificity. Here, we propose that the Kraken classifier coupled with a custom Kraken database comprising plasmid-free chromosomal sequences and complete plasmid sequences can be used for detection of plasmid contigs in draft genome assemblies. We showed that Kraken achieved balanced and higher performance compared with other methods (Centrifuge, RFPlasmid, mlplasmids, PlaScope, and Platon). We therefore consider that the Kraken classifier can be the best option for predicting the origin of contigs for species with a suitable number of completed chromosomal and plasmid sequences.

scholarly journals Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 598
Author(s):  
Katharina Juraschek ◽  
Maria Borowiak ◽  
Simon H. Tausch ◽  
Burkhard Malorny ◽  
Annemarie Käsbohrer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Mobile Genetic Elements ◽  
Hybrid Assembly ◽  
Small Plasmid ◽  
Short Read ◽  
Short Read Sequencing ◽  
Genetic Elements ◽  
Antimicrobial Resistance Genes ◽  
Long Read

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E.coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.

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