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).
Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the ...asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period.
We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks.
A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period.
Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways.
ClinicalTrials.gov NCT02671773.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The aim of this study was to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data to quantify genetic variation to assess within-outbreak strain relatedness and characterise ...microevolutionary events in the accessory genomes of a cluster of 23 genetically and epidemiologically linked isolates related to an outbreak of Shiga toxin-producing Escherichia coli O157:H7 caused by the consumption of raw drinking milk. There were seven discrepant variants called between the two technologies, five were false-negative or false-positive variants in the Illumina data and two were false-negative calls in ONT data. After masking horizontally acquired sequences such as prophages, analysis of both short and long-read sequences revealed the 20 isolates linked to the outbreak in 2017 had a maximum SNP distance of one SNP between each other, and a maximum of five SNPs when including three additional strains identified in 2019. Analysis of the ONT data revealed a 47 kbp deletion event in a terminal compound prophage within one sample relative to the remaining samples, and a 0.65 Mbp large chromosomal rearrangement (inversion), within one sample relative to the remaining samples. Furthermore, we detected two bacteriophages encoding the highly pathogenic Shiga toxin (Stx) subtype, Stx2a. One was typical of Stx2a-phage in this sub-lineage (Ic), the other was atypical and inserted into a site usually occupied by Stx2c-encoding phage. Finally, we observed an increase in the size of the pO157 IncFIB plasmid (1.6 kbp) in isolates from 2019 compared to those from 2017, due to the duplication of insertion elements within the plasmids from the more recently isolated strains. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the genome plasticity and virulence between strains of this zoonotic, foodborne pathogen.
Propionibacterium acnes subsp. acnes subsp. nov. and Propionibacterium acnes subsp. elongatum subsp. nov. are described. These emanate from the three known phylotypes of P. acnes, designated types I, ...II and III. Electron microscopy confirmed the filamentous cell shape of type III, showing a striking difference from types I/II, which were short rods. Biochemical tests indicated that, in types I/II, either the pyruvate, l-pyrrolidonyl arylamidase or d-ribose 2 test was positive, whereas all of these were negative among type III strains. Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, which profile mainly their ribosomal proteins, were different between these two groups. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) spectra of all phylotypes revealed a specific protein biomarker that was overexpressed in type III strains compared with types I/II only when grown aerobically. Reference strains had high whole-genome similarity between types I (>91 %) and II (>75 %), but a considerably lower level of 72 % similarity with type III. recA and gyrB sequence dendrograms confirmed the distant relatedness of type III, indicating the presence of two distinct centres of variation within the species P. acnes. On the other hand, cellular fatty acid profiles and 16S rRNA gene sequence relatedness (>99.3 %) circumscribed the species. Thus, we propose two subspecies, Propionibacterium acnes subsp. acnes subsp. nov. for types I/II and Propionibacterium acnes subsp. elongatum subsp. nov. for type III. The type strain of Propionibacterium acnes subsp. acnes is NCTC 737T ( = ATCC 6919T = JCM 6425T = DSM 1897T = CCUG 1794T), while the type strain of Propionibacterium acnes subsp. elongatum is K124T ( = NCTC 13655T = JCM 18919T).
Sequence similarity of pathogen genomes can infer the relatedness between isolates as the fewer genetic differences identified between pairs of isolates, the less time since divergence from a common ...ancestor. Clustering based on hierarchical single linkage clustering of pairwise SNP distances has been employed to detect and investigate outbreaks. Here, we evaluated the evidence-base for the interpretation of phylogenetic clusters of Shiga toxin-producing
(STEC) O157:H7. Whole genome sequences of 1193 isolates of STEC O157:H7 submitted to Public Health England between July 2015 and December 2016 were mapped to the Sakai reference strain. Hierarchical single linkage clustering was performed on the pairwise SNP difference between all isolates at descending distance thresholds. Cases with known epidemiological links fell within 5-SNP single linkage clusters. Five-SNP single linkage community clusters where an epidemiological link was not identified were more likely to be temporally and/or geographically related than sporadic cases. Ten-SNP single linkage clusters occurred infrequently and were challenging to investigate as cases were few, and temporally and/or geographically dispersed. A single linkage cluster threshold of 5-SNPs has utility for the detection of outbreaks linked to both persistent and point sources. Deeper phylogenetic analysis revealed that the distinction between domestic UK and imported isolates could be inferred at the sub-lineage level. Cases associated with domestically acquired infection that fall within clusters that are predominantly travel associated are likely to be caused by contaminated imported food.
Increasing levels of antimicrobial resistance (AMR) have been documented in
Escherichia coli
causing travellers’ diarrhoea, particularly to the third-generation cephalosporins. Diarrhoeagenic
E. coli
...(DEC) can act as a reservoir for the exchange of AMR genes between bacteria residing in the human gut, enabling them to survive and flourish through the selective pressures of antibiotic treatments. Using Oxford Nanopore Technology (ONT), we sequenced eight isolates of DEC from four patients’ specimens who had all recently returned to the United Kingdome from Pakistan. Sequencing yielded two DEC harbouring
bla
CTX-M-15
per patient, all with different sequence types (ST) and belonging to five different pathotypes. The study aimed to determine whether
bla
CTX-M-15
was located on the chromosome or plasmid and to characterise the drug-resistant regions to better understand the mechanisms of onward transmission of AMR determinants. Patients A and C both had one isolate where
bla
CTX-M-15
was located on the plasmid (899037 & 623213, respectively) and one chromosomally encoded (899091 & 623214, respectively). In patient B,
bla
CTX-M-15
was plasmid-encoded in both DEC isolates (786605 & 7883090), whereas in patient D,
bla
CTX-M-15
was located on the chromosome in both DEC isolates (542093 & 542099). The two
bla
CTX-M-15
-encoding plasmids associated with patient B were different although the
bla
CTX-M-15
-encoding plasmid isolated from 788309 (IncFIB) exhibited high nucleotide similarity to the
bla
CTX-M-15
-encoding plasmid isolated from 899037 (patient A). In the four isolates where
bla
CTX-M-15
was chromosomally encoded, two isolates (899091 & 542099) shared the same insertion site. The
bla
CTX-M-15
insertion site in isolate 623214 was described previously, whereas that of isolate 542093 was unique to this study. Analysis of Nanopore sequencing data enables us to characterise the genomic architecture of mobile genetic elements encoding AMR determinants. These data may contribute to a better understanding of persistence and onward transmission of AMR determinants in multidrug-resistant (MDR)
E. coli
causing gastrointestinal and extra-intestinal infections.
The increasing use of PCR for the detection of gastrointestinal pathogens in hospital laboratories in England has improved the detection of Shiga toxin-producing
(STEC), and the diagnosis of ...haemolytic uraemic syndrome (HUS). We aimed to analyse the microbiological characteristics and phylogenetic relationships of STEC O26:H11, clonal complex (CC) 29, in England to inform surveillance, and to assess the threat to public health. There were 502 STEC belonging to CC29 isolated between 2014 and 2019, of which 416 were from individual cases. The majority of isolates belonged to one of three major sequence types (STs), ST16 (
=37), ST21 (
=350) and ST29 (
=24). ST16 and ST29 were mainly isolated from cases reporting recent travel abroad. Within ST21, there were three main clades associated with domestic acquisition. All three domestic clades had Shiga toxin subtype gene (
) profiles associated with causing severe clinical outcomes including STEC-HUS, specifically either
,
or
. Isolates from the same patient, same household or same outbreak with an established source for the most part fell within 5-SNP single linkage clusters. There were 19 5-SNP community clusters, of which six were travel-associated and one was an outbreak of 16 cases caused by the consumption of contaminated salad leaves. Of the remaining 12 clusters, 9/12 were either temporally or geographically related or both. Exposure to foodborne STEC O26:H11 ST21 capable of causing severe clinical outcomes, including STEC-HUS, is an emerging risk to public health in England. The lack of comprehensive surveillance of this STEC serotype is a concern, and there is a need to expand the implementation of methods capable of detecting STEC in local hospital settings.
Spoligotyping is a tool for the molecular characterization/typing of Mycobacterium tuberculosis complex (MTBC) strains based on target sequences (spacers) in the direct repeat (DR) region (14). The ...standard spoligotyping assay involves the hybridization of amplified sample DNA to nylon membrane-immobilized oligonucleotides whose sequences are representative of 43 spacer regions. Variations in the number of spacers as a result of deletions of adjacent blocks of repetitive units allow the differentiation of clinical isolates. In the present study, we developed a new multiplexed primer extension-based spoligotyping assay using automated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that improves the classical reverse line blot hybridization assay with respect to reproducibility, throughput, process flow, ease of use, and data analysis. Validation of the MALDI-TOF MS-based spoligotyping assay with two sample sets with a total of 326 samples resulted in 96.6% concordance (315/326) when the full spoligotype patterns were compared with the results of standard spoligotyping and 99.9% concordance when the results were compared with those of individual primer extension assays. Ten strains (including two Mycobacterium canettii strains) showed discordant results with one or two spacer differences from the membrane-based spoligotyping result. Most discordant samples were identified to be the result of ambiguities in the interpretation of weak hybridization signals in the reverse line blot assay and sequence variations in the spacer regions. We established a new automated primer extension assay and successfully validated it for use for the routine typing of MTBC strains in the research and public health laboratory environments. The present multiplex levels of up to 30 are extendable and allow the additional incorporation of controls and antibiotic resistance markers.
Traditional microbial typing technologies for the characterization of pathogenic microorganisms and monitoring of their global spread are often difficult to standardize and poorly portable, and they ...lack sufficient ease of use, throughput, and automation. To overcome these problems, we introduce the use of comparative sequencing by MALDI-TOF MS for automated high-throughput microbial DNA sequence analysis. Data derived from the public multilocus sequence typing (MLST) database (http://pubmlst.org/neisseria) established a reference set of expected peak patterns. A model pathogen, Neisseria meningitidis, was used to validate the technology and explore its applicability as an alternative to dideoxy sequencing. One hundred N. meningitidis samples were typed by comparing MALDI-TOF MS fingerprints of the standard MLST loci to reference sequences available in the public MLST database. Identification results can be obtained in 2 working days. Results were in concordance with classical dideoxy sequencing with 98% correct automatic identification. Sequence types (STs) of 89 samples were represented in the database, seven samples revealed new STs, including three new alleles, and four samples contained mixed populations of multiple STs. The approach shows interlaboratory reproducibility and allows for the exchange of mass spectrometric fingerprints to study the geographic spread of epidemic N. meningitidis strains or other microbes of clinical importance.
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