The increasing availability and decreasing cost of high-throughput sequencing has transformed academic medical microbiology, delivering an explosion in available genomes while also driving advances ...in bioinformatics. However, many microbiologists are unable to exploit the resulting large genomics datasets because they do not have access to relevant computational resources and to an appropriate bioinformatics infrastructure. Here, we present the Cloud Infrastructure for Microbial Bioinformatics (CLIMB) facility, a shared computing infrastructure that has been designed from the ground up to provide an environment where microbiologists can share and reuse methods and data.
In October 2015, a new case of Ebola virus disease in Guinea was detected. Case investigation, serology, and whole-genome sequencing indicated possible transmission of the virus from an Ebola virus ...disease survivor to another person and then to the case-patient reported here. This transmission chain over 11 months suggests slow Ebola virus evolution.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) now arise in the context of heterogeneous human connectivity and population immunity. Through a large-scale ...phylodynamic analysis of 115,622 Omicron BA.1 genomes, we identified >6,000 introductions of the antigenically distinct VOC into England and analyzed their local transmission and dispersal history. We find that six of the eight largest English Omicron lineages were already transmitting when Omicron was first reported in southern Africa (22 November 2021). Multiple datasets show that importation of Omicron continued despite subsequent restrictions on travel from southern Africa as a result of export from well-connected secondary locations. Initiation and dispersal of Omicron transmission lineages in England was a two-stage process that can be explained by models of the country's human geography and hierarchical travel network. Our results enable a comparison of the processes that drive the invasion of Omicron and other VOCs across multiple spatial scales.
Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and bacteremia, estimated to cause 2 million deaths annually. The majority of pneumococcal mortality occurs in developing ...countries, with serotype 1 a leading cause in these areas. To begin to better understand the larger impact that serotype 1 strains have in developing countries, we characterized virulence and genetic content of PNI0373, a serotype 1 strain from a diseased patient in The Gambia. PNI0373 and another African serotype 1 strain showed high virulence in a mouse intraperitoneal challenge model, with 20% survival at a dose of 1 cfu. The PNI0373 genome sequence was similar in structure to other pneumococci, with the exception of a 100 kb inversion. PNI0373 showed only 15 lineage specific CDS when compared to the pan-genome of pneumococcus. However analysis of non-core orthologs of pneumococcal genomes, showed serotype 1 strains to be closely related. Three regions were found to be serotype 1 associated and likely products of horizontal gene transfer. A detailed inventory of known virulence factors showed that some functions associated with colonization were absent, consistent with the observation that carriage of this highly virulent serotype is unusual. The African serotype 1 strains thus appear to be closely related to each other and different from other pneumococci despite similar genetic content.
Summary
Laboratory‐based evolution and whole‐genome sequencing can link genotype and phenotype. We used evolution of acid resistance in exponential phase Escherichia coli to study resistance to a ...lethal stress. Iterative selection at pH 2.5 generated five populations that were resistant to low pH in early exponential phase. Genome sequencing revealed multiple mutations, but the only gene mutated in all strains was evgS, part of a two‐component system that has already been implicated in acid resistance. All these mutations were in the cytoplasmic PAS domain of EvgS, and were shown to be solely responsible for the resistant phenotype, causing strong upregulation at neutral pH of genes normally induced by low pH. Resistance to pH 2.5 in these strains did not require the transporter GadC, or the sigma factor RpoS. We found that EvgS‐dependent constitutive acid resistance to pH 2.5 was retained in the absence of the regulators GadE or YdeO, but was lost if the oxidoreductase YdeP was also absent. A deletion in the periplasmic domain of EvgS abolished the response to low pH, but not the activity of the constitutive mutants. On the basis of these results we propose a model for how EvgS may become activated by low pH.
We conducted the genome sequencing and analysis of the first confirmed COVID-19 infections in Brazil. Rapid sequencing coupled with phylogenetic analyses in the context of travel history corroborate ...multiple independent importations from Italy and local spread during the initial stage of COVID-19 transmission in Brazil.
FNDR-20081 4-{4-5-(4-Isopropyl-phenyl)- 1,2,4oxadiazol-3-ylmethyl-piperazin-1-yl}-7-pyridin-3-yl-quinoline is a novel, first in class anti-tubercular pre-clinical candidate against sensitive and ...drug-resistant Mycobacterium tuberculosis (Mtb). In-vitro combination studies of FNDR-20081 with first- and second-line drugs exhibited no antagonism, suggesting its compatibility for developing new combination-regimens. FNDR-20081, which is non-toxic with no CYP3A4 liability, demonstrated exposure-dependent killing of replicating-Mtb, as well as the non-replicating-Mtb, and efficacy in a mouse model of infection. Whole genome sequencing (WGS) of FNDR-20081 resistant mutants revealed the identification of pleotropic targets: marR (Rv0678), a regulator of MmpL5, a transporter/efflux pump mechanism for drug resistance; and Rv3683, a putative metalloprotease potentially involved in peptidoglycan biosynthesis. In summary, FNDR-20081 is a promising first in class compound with the potential to form a new combination regimen for MDR-TB treatment.
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Objectives Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa ...after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition. Study design An observational prospective cohort study. Setting Burns care ward and critical care ward in the UK. Participants Patients with >7% total burns by surface area were recruited into the study. Methods All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates. Results WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment. Conclusions This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward.
Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore ...sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis.
Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix
SK-2S, Sugi
Eyespear, Cotton, Rayon, Dryswab
, Hydraflock
, Albumin-coated, Purflock
, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods.
We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (
< 0.001), with the Sugi
Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results.
We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.
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