Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest ...set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Horizontal gene transfer has played a role in developing the global public health crisis of antimicrobial resistance (AMR). However, the dynamics of AMR transfer through bacterial populations and its ...direct impact on human disease is poorly elucidated. Here, we study parallel epidemic emergences of multiple Shigella species, a priority AMR organism, in men who have sex with men to gain insight into AMR emergence and spread. Using genomic epidemiology, we show that repeated horizontal transfer of a single AMR plasmid among Shigella enhanced existing and facilitated new epidemics. These epidemic patterns contrasted with slighter, slower increases in disease caused by organisms with vertically inherited (chromosomally encoded) AMR. This demonstrates that horizontal transfer of AMR directly affects epidemiological outcomes of globally important AMR pathogens and highlights the need for integration of genomic analyses into all areas of AMR research, surveillance and management.
Multi-country outbreaks of foodborne bacterial disease present challenges in their detection, tracking, and notification. As food is increasingly distributed across borders, such outbreaks are ...becoming more common. This increases the need for high-resolution, accessible, and replicable isolate typing schemes. Here we evaluate a core genome multilocus typing (cgMLST) scheme for the high-resolution reproducible typing of Salmonella enterica (S. enterica) isolates, by its application to a large European outbreak of S. enterica serovar Enteritidis. This outbreak had been extensively characterised using single nucleotide polymorphism (SNP)-based approaches. The cgMLST analysis was congruent with the original SNP-based analysis, the epidemiological data, and whole genome MLST (wgMLST) analysis. Combination of the cgMLST and epidemiological data confirmed that the genetic diversity among the isolates predated the outbreak, and was likely present at the infection source. There was consequently no link between country of isolation and genetic diversity, but the cgMLST clusters were congruent with date of isolation. Furthermore, comparison with publicly available Enteritidis isolate data demonstrated that the cgMLST scheme presented is highly scalable, enabling outbreaks to be contextualised within the Salmonella genus. The cgMLST scheme is therefore shown to be a standardised and scalable typing method, which allows Salmonella outbreaks to be analysed and compared across laboratories and jurisdictions.
•cgMLST is proposed as a universal typing scheme for Salmonella.•cgMLST is congruent with SNP analyses and easier to implement across laboratories.•Genomic data are consistent with the epidemiology of the outbreak.
Salmonella enterica serotype Typhimurium (S. Typhimurium) is a leading cause of gastroenteritis and bacteraemia worldwide, and a model organism for the study of host-pathogen interactions. Two S. ...Typhimurium strains (SL1344 and ATCC14028) are widely used to study host-pathogen interactions, yet genotypic variation results in strains with diverse host range, pathogenicity and risk to food safety. The population structure of diverse strains of S. Typhimurium revealed a major phylogroup of predominantly sequence type 19 (ST19) and a minor phylogroup of ST36. The major phylogroup had a population structure with two high order clades (α and β) and multiple subclades on extended internal branches, that exhibited distinct signatures of host adaptation and anthropogenic selection. Clade α contained a number of subclades composed of strains from well characterized epidemics in domesticated animals, while clade β contained multiple subclades associated with wild avian species. The contrasting epidemiology of strains in clade α and β was reflected by the distinct distribution of antimicrobial resistance (AMR) genes, accumulation of hypothetically disrupted coding sequences (HDCS), and signatures of functional diversification. These observations were consistent with elevated anthropogenic selection of clade α lineages from adaptation to circulation in populations of domesticated livestock, and the predisposition of clade β lineages to undergo adaptation to an invasive lifestyle by a process of convergent evolution with of host adapted Salmonella serotypes. Gene flux was predominantly driven by acquisition and recombination of prophage and associated cargo genes, with only occasional loss of these elements. The acquisition of large chromosomally-encoded genetic islands was limited, but notably, a feature of two recent pandemic clones (DT104 and monophasic S. Typhimurium ST34) of clade α (SGI-1 and SGI-4).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 ...patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella.
We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h.
Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices.
Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a systemic human infection with a burden exceeding 20 million cases each year that occurs disproportionately ...among children in low and middle income countries. Antimicrobial therapy is the mainstay for treatment, but resistance to multiple agents is common. Here we report genotypes and antimicrobial resistance (AMR) determinants detected from routine whole-genome sequencing (WGS) of 533 S. Typhi isolates referred to Public Health England between April 2014 and March 2017, 488 (92%) of which had accompanying patient travel information obtained via an enhanced surveillance questionnaire. The majority of cases involved S. Typhi 4.3.1 (H58) linked with travel to South Asia (59%). Travel to East and West Africa were associated with genotypes 4.3.1 and 3.3.1, respectively. Point mutations in the quinolone resistance determining region (QRDR), associated with reduced susceptibility to fluoroquinolones, were very common (85% of all cases) but the frequency varied significantly by region of travel: 95% in South Asia, 43% in East Africa, 27% in West Africa. QRDR triple mutants, resistant to ciprofloxacin, were restricted to 4.3.1 lineage II and associated with travel to India, accounting for 23% of cases reporting travel to the country. Overall 24% of isolates were MDR, however the frequency varied significantly by region and country of travel: 27% in West Africa, 52% in East Africa, 55% in Pakistan, 24% in Bangladesh, 3% in India. MDR determinants were plasmid-borne (IncHI1 PST2 plasmids) in S. Typhi 3.1.1 linked to West Africa, but in all other regions MDR was chromosomally integrated in 4.3.1 lineage I. We propose that routine WGS data from travel-associated cases in industrialised countries could serve as informal sentinel AMR genomic surveillance data for countries where WGS is not available or routinely performed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Shigella are globally important diarrhoeal pathogens that are endemic in low-to-middle income nations and also occur in high income nations, typically in travellers or community-based risk-groups. ...Shigella phylogenetics reveals population structures that are more reliable than those built with traditional typing methods, and has identified sublineages associated with specific geographical regions or patient groups. Genomic analyses reveal temporal increases in Shigella antimicrobial resistance (AMR) gene content, which is frequently encoded on mobile genetic elements. Here, we whole genome sequenced representative subsamples of S. flexneri 2a and S. sonnei (n = 366) from the United Kingdom from 2008 to 2014, and analysed these alongside publicly available data to make qualitative insights on the genomic epidemiology of shigellosis and its AMR within the broader global context. Combined phylogenetic, epidemiological and genomic anlayses revealed the presence of domestically-circulating sublineages in patient risk-groups and the importation of travel-related sublineages from both Africa and Asia, including ciprofloxacin-resistant sublineages of both species from Asia. Genomic analyses revealed common AMR determinants among travel-related and domestically-acquired isolates, and the evolution of mutations associated with reduced quinolone susceptibility in domestically-circulating sublineages. Collectively, this study provides unprecedented insights on the contribution and mobility of endemic and travel-imported sublineages and AMR determinants responsible for disease in a high-income nation.
Antibiotic resistance is a major problem in
serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with ...the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel
Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR
Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the
extended-spectrum β-lactamase, and carrying the
fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of
Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.
Typhoid fever is a severe disease caused by the Gram-negative bacterium
serovar Typhi. Antibiotic-resistant
Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR)
Typhi clone in Sindh, Pakistan. The XDR
Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in
Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR
Typhi to investigate the phylogenetic relationship between these isolates and a global collection of
Typhi isolates and to identify multiple genes linked to antibiotic resistance. This
Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in
Typhi observed here adds urgency to the need for typhoid prevention measures.
Escherichia coli and Shigella species are closely related and genetically constitute the same species. Differentiating between these two pathogens and accurately identifying the four species of ...Shigella are therefore challenging. The organism-specific bioinformatics whole-genome sequencing (WGS) typing pipelines at Public Health England are dependent on the initial identification of the bacterial species by use of a kmer-based approach. Of the 1,982 Escherichia coli and Shigella sp. isolates analyzed in this study, 1,957 (98.4%) had concordant results by both traditional biochemistry and serology (TB&S) and the kmer identification (ID) derived from the WGS data. Of the 25 mismatches identified, 10 were enteroinvasive E. coli isolates that were misidentified as Shigella flexneri or S. boydii by the kmer ID, and 8 were S. flexneri isolates misidentified by TB&S as S. boydii due to nonfunctional S. flexneri O antigen biosynthesis genes. Analysis of the population structure based on multilocus sequence typing (MLST) data derived from the WGS data showed that the remaining discrepant results belonged to clonal complex 288 (CC288), comprising both S. boydii and S. dysenteriae strains. Mismatches between the TB&S and kmer ID results were explained by the close phylogenetic relationship between the two species and were resolved with reference to the MLST data. Shigella can be differentiated from E. coli and accurately identified to the species level by use of kmer comparisons and MLST. Analysis of the WGS data provided explanations for the discordant results between TB&S and WGS data, revealed the true phylogenetic relationships between different species of Shigella, and identified emerging pathoadapted lineages.
Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly ...common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring the spread of resistant clones, including those that have recently emerged, at local and global scales.
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