ORCID Profile
0000-0001-7470-3786
Current Organisation
University of Nottingham
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Publisher: Elsevier BV
Date: 02-2010
DOI: 10.1016/J.IJMM.2009.08.015
Abstract: Staphylococcus aureus has a formidable ability to adapt to varying environmental conditions and an extraordinary capacity to rapidly become resistant to virtually all antibiotics. Resistance develops either through mutations and rearrangements within the staphylococcal genome, or by the acquisition of resistance determinants. Antibiotic resistances often impose a fitness burden on the host. Such biological costs can be reduced by tight regulation and antibiotic-inducible expression of resistance genes, or by compensatory mutations. Resistance induction by antibiotics can be mediated by dedicated, antibiotic-recognizing signal transducers or by mechanisms relieving translational attenuation. Antibiotic tolerance and the expression of resistance phenotypes can also be strongly influenced by the genetic backgrounds of strains and several other factors. Modification and indirect regulation of resistance levels can occur by mutations that alter gene expression or substrate specificity of genes contributing to resistance. Insertion elements can alter resistance profiles by turning relevant genes on or off. Environmental conditions and stress response mechanisms triggered by perturbation of the cell envelope, DNA damage, or faulty intermediary metabolism can also have an impact on resistance development and expression. Clinically relevant resistance is often built up through multiple steps, each of which contributes to an increase in resistance. The driving force behind resistance formation is antibiotic stress, and under clinical conditions selection for resistance is continuously competing with selection for bacterial fitness.
Publisher: American Society for Microbiology
Date: 11-2007
DOI: 10.1128/AAC.00722-07
Abstract: The inactivation of TcaA contributes to intrinsic teicoplanin resistance in experimental and clinical isolates of glycopeptide-intermediate resistant Staphylococcus aureus . PhoA fusions confirmed that TcaA is a transmembrane protein with a short intracellular N-terminal domain containing a C-4 zinc finger binding motif, a single membrane-spanning domain, and a large extracellular C-terminal domain. The region conferring teicoplanin susceptibility was narrowed down to the transmembrane part and the first third of the extracellular domain of TcaA, suggesting that neither the C-4 zinc finger binding motif nor the C terminus contributed to teicoplanin susceptibility. TcaA belongs to the cell wall stress stimulon, which comprises a set of genes universally upregulated by cell wall damage. Induction of tcaA was shown to be fully dependent on the two-component regulatory system VraSR. A 66-bp region upstream of the transcriptional start site, which contained an inverted repeat partially covering the promoter box, was shown to be essential for VraSR-mediated induction by cell wall stress. Interestingly, the induction or overexpression of tcaA did not contribute further to teicoplanin susceptibility, suggesting that small amounts of TcaA, such as those present under normal uninduced conditions, were sufficient for TcaA-mediated teicoplanin susceptibility. The strong attenuation of tcaA deletion mutants in a Caenorhabditis elegans survival assay suggested that TcaA may, in addition to affecting glycopeptide susceptibility, also play a role in virulence.
Publisher: Oxford University Press (OUP)
Date: 14-05-2015
DOI: 10.1093/JAC/DKV128
Abstract: Detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates presents significant challenges in settings with no dominant clonal lineages, such as Australia. We assessed the utility of WGS versus standard PCR lification assays for the characterization of pyrazinamide resistance in MDR-TB isolates identified in New South Wales, Australia, over an 8 year period. PCR licon sequencing was used to identify molecular markers associated with antibiotic resistance in pyrazinamide-resistant MDR-TB isolates recovered by the New South Wales Mycobacterium Reference Laboratory between 2007 and 2014. WGS was subsequently performed on two isolates for which pncA lification failed. WGS identified two novel genomic deletions associated with in vitro resistance to pyrazinamide in MDR-TB. One isolate also carried a second deletion involving the genes dfrA and thyA associated with resistance to para-aminosalicylic acid. Steadily decreasing sequencing costs are increasing the appeal of WGS as an alternative approach for detecting complex patterns of pyrazinamide resistance in MDR-TB.
Publisher: Wiley
Date: 05-07-2004
Publisher: Springer Science and Business Media LLC
Date: 02-07-2007
Abstract: An extremely low level methicillin resistant Staphylococcus aureus (MRSA) belonging to ST45, circulates among intravenous drug users in the Zurich area. This clone can be misinterpreted as an MSSA by phenotypic oxacillin resistance tests, although it carries a staphylococcal cassette chromosome mec (SCC mec ) element encoding a functional mecA gene and it produces PBP2a. This clone carried a new 45.7-kb element, termed SCC mec N1 , containing a class B mec complex ( mecA- Δ mecR1::IS1272 ), a truncated Tn 4003 harbouring the dfrA gene, and a fusB1 gene, conferring methicillin, trimethoprim and low level fusidic acid resistance, respectively. In addition to the two insertion site sequences (ISS) framing the SCC mec , a third ISS (ISS*) was identified within the element. SCC mec N1 also harboured two distinct ccrAB complexes belonging to the class 4 subtype, both of which were shown to be active and to be able to excise the SCC mec N1 or parts thereof. Slight variations in the SmaI-PFGE pattern of the clinical MRSA isolates belonging to this clone were traced back to differences in the sizes of the SCC mec J2 regions and/or to a 6.4-kb deletion extending from ISS* to the right end ISS. This latter deletion led to a variant right SCC mec -chromosomal junction site. MRSA clones carrying the shorter SCC mec with the 6.4-kb deletion were usually ciprofloxacin resistant, while strains with the complete SCC mec N1 were co-trimoxazole resistant or had no additional resistances. This suggested that the genetic backbone of the host S. aureus , although identical by PFGE pattern, had at some stage erged with one branch acquiring a sulfonomide resistance mutation and the other ciprofloxacin resistance. This description of the structure and variations of SCC mec N1 will allow for quicker and easier molecular detection of this clone and monitoring of its spread.
Publisher: Springer Science and Business Media LLC
Date: 26-11-2008
DOI: 10.1007/S10096-008-0663-7
Abstract: A periodic survey of methicillin-resistant Staphylococcus aureus (MRSA) in Zurich in 2004 and 2006 revealed a consistently low prevalence of MRSA. SCCmec and ccr typing showed fluctuations in the proportions of SCCmec types and in the carriage of mobile virulence determinants. Together with the presence of variant SCCmecs these findings suggest a high clonal ersity and level of SCCmec recombination. The prevalence of a local "drug clone", associated with low-level methicillin resistance and rapid growth, significantly decreased. This clone had spread among intraveneous drug users, steadily increasing from 1994 to 2001 and was dominant in 2001. Apparently, changes in the management of the Zurich drug scene have restricted the spread of this clone.
Publisher: American Society for Microbiology
Date: 2007
DOI: 10.1128/AAC.00921-06
Abstract: A novel staphylococcal cassette chromosome (SCC) mec from a clinical methicillin-resistant Staphylococcus aureus isolate (ST100/CC5) had a mosaic structure, composed of SCC DNA from several different backgrounds. It harbored two complete ccr loci and a new variant of mec complex B, with Δ mecR1 interrupted by the aminoglycoside resistance transposon Tn 4001 .
Publisher: Springer Science and Business Media LLC
Date: 18-11-2019
Publisher: Microbiology Society
Date: 06-2021
DOI: 10.1099/JGV.0.001595
Abstract: In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on in iduals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected in idual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea – also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger s le of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.
Publisher: American Society for Microbiology
Date: 06-2004
DOI: 10.1128/AAC.48.6.1953-1959.2004
Abstract: The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus . By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS 256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.
Publisher: American Society for Microbiology
Date: 09-2015
DOI: 10.1128/JCM.00143-15
Abstract: Infant botulism is a potentially life-threatening paralytic disease that can be associated with prolonged morbidity if not rapidly diagnosed and treated. Four infants were diagnosed and treated for infant botulism in NSW, Australia, between May 2011 and August 2013. Despite the temporal relationship between the cases, there was no close geographical clustering or other epidemiological links. Clostridium botulinum isolates, three of which produced botulism neurotoxin serotype A (BoNT/A) and one BoNT serotype B (BoNT/B), were characterized using whole-genome sequencing (WGS). In silico multilocus sequence typing (MLST) found that two of the BoNT/A-producing isolates shared an identical novel sequence type, ST84. The other two isolates were single-locus variants of this sequence type (ST85 and ST86). All BoNT/A-producing isolates contained the same chromosomally integrated BoNT/A2 neurotoxin gene cluster. The BoNT/B-producing isolate carried a single plasmid-borne bont /B gene cluster, encoding BoNT subtype B6. Single nucleotide polymorphism (SNP)-based typing results corresponded well with MLST however, the extra resolution provided by the whole-genome SNP comparisons showed that the isolates differed from each other by ,500 SNPs. WGS analyses indicated that the four infant botulism cases were caused by genomically distinct strains of C. botulinum that were unlikely to have originated from a common environmental source. The isolates did, however, cluster together, compared with international isolates, suggesting that C. botulinum from environmental reservoirs throughout NSW have descended from a common ancestor. Analyses showed that the high resolution of WGS provided important phylogenetic information that would not be captured by standard seven-loci MLST.
Publisher: Cold Spring Harbor Laboratory
Date: 16-08-2019
DOI: 10.1101/735928
Abstract: After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2 . The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2 , along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the ∼2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis liconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.
Publisher: Springer Science and Business Media LLC
Date: 14-07-2020
DOI: 10.1038/S41586-020-2547-7
Abstract: After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2 . Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis liconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
Publisher: Springer Science and Business Media LLC
Date: 15-09-2016
Publisher: American Society for Microbiology
Date: 15-05-2004
DOI: 10.1128/JB.186.10.2966-2972.2004
Abstract: TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel Staphylococcus aureus regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that TcaR upregulates sarS and thus spa transcription. In addition, it attenuates whole-length transcription of sasF , thereby producing a truncated transcript lacking the 3′ terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact tcaR gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of tcaR , harboring an inactive TcaR and therefore expressing very low levels of sarS . The data presented here identified TcaR as a further activator of sarS , and a modulator of sasF expression that has to be taken into account in studies of virulence gene expression in S. aureus .
Publisher: Public Library of Science (PLoS)
Date: 13-10-2016
Publisher: Public Library of Science (PLoS)
Date: 27-08-2013
Publisher: Elsevier BV
Date: 05-2006
DOI: 10.1016/J.INJURY.2006.04.005
Abstract: Bacteria have adapted a variety of different ways to acquire antibiotic resistance, fostering the rapid development of resistance within a short evolutionary time. The general genetic basis of events leading to and promoting antibiotic resistance formation in bacteria are presented and exemplified by showing the evolution of methicillin, glycopeptide, linezolid, and ketolide resistance in Staphylococcus aureus.
Publisher: Springer Science and Business Media LLC
Date: 20-01-2011
Abstract: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by erse cell wall active agents. We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 ( S. aureus N315), which detects very subtle differences in expression as well as measuring 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes. CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.
Publisher: American Society for Microbiology
Date: 06-2004
DOI: 10.1128/AAC.48.6.2295-2297.2004
Abstract: Transformation of a type I SCC mec element into Staphylococcus aureus yielded highly oxacillin-resistant transformants with a reduced growth rate. Faster-growing variants could again be selected at the cost of reduced resistance levels, demonstrating an inverse correlation between oxacillin resistance levels and growth rate.
Publisher: Springer New York
Date: 2016
DOI: 10.1007/978-1-4939-3676-2_11
Abstract: Luciferase reporter gene fusions provide an extremely rapid and sensitive tool for measuring the induction or repression of stress responses in bacteria. Staphylococcus aureus activates the expression of a cell wall stress stimulon (CWSS) in response to the inhibition or disruption of cell wall synthesis. The highly sensitive promoter-reporter gene fusion construct psas016 p-luc+ can be used to quantify and compare any changes in CWSS expression levels and induction kinetics. Potential uses of this system include identifying and characterizing novel cell wall-targeting antibacterial agents, identifying genomic loci influencing cell envelope synthesis and detecting changes in CWSS expression that could be linked to decreased antibiotic susceptibility profiles in clinical isolates.
Publisher: American Society for Microbiology
Date: 06-2002
DOI: 10.1128/JB.184.11.3086-3095.2002
Abstract: The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.
Publisher: Elsevier BV
Date: 10-2023
Publisher: American Society for Microbiology
Date: 04-2011
DOI: 10.1128/AAC.01213-10
Abstract: The exposure of Staphylococcus aureus to a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. The vraSR genes form part of the four-cistron autoregulatory operon orf1-yvqF-vraS-vraR . The markerless inactivation of each of the genes within this operon revealed that orf1 played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF in S. aureus appears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.
Publisher: Cold Spring Harbor Laboratory
Date: 21-08-2020
DOI: 10.1101/2020.08.18.20174623
Abstract: In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on in iduals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected in idual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea – also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger s le of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.PATHOL.2017.10.014
Abstract: Bordetella pertussis, the aetiological agent of whooping cough is routinely diagnosed by polymerase chain reaction (PCR) directed at IS481, an insertion sequence target also found in Bordetella holmesii. Recent reports have suggested that B. holmesii infections can be misdiagnosed as pertussis, which can have a significant impact on public health surveillance. This study investigated the presence of B. holmesii in B. pertussis positive clinical s les, in order to determine the incidence of B. holmesii. Clinical cases of pertussis diagnosed by IS481-specific PCR between October 2008 and March 2016 in New South Wales were included. Bordetella holmesii was detected through the simultaneous lification of IS481 and B. holmesii specific insertions sequence, hIS1001. A total of 46 of 802 patients were identified to be positive for B. holmesii rather than B. pertussis, suggesting an incidence rate of 6.5% in 2009, 16.8% in 2010, 7.6% during 2013 and 8.1% during 2015. Bordetella holmesii infections were diagnosed during and between pertussis epidemics, however cases of B. holmesii and B. pertussis co-infections were not found. The predominant age group of B. holmesii infection was 11-18 years old, which was significantly different to the mean age of B. pertussis infections (0-6 years, p = 0.023). These findings revealed that B. holmesii was co-circulating alongside the B. pertussis epidemic for seven years, hidden from view, as B. holmesii infections have been diagnosed as B. pertussis. Confirmatory testing of B. pertussis positive s les for the presence of B. holmesii, especially during pertussis epidemics, should improve the quality of laboratory diagnosis and laboratory surveillance for pertussis. The presence of B. holmesii in Australia highlights the importance of testing for this pathogen and ongoing molecular surveillance that can guide the control of whooping cough.
Publisher: Cold Spring Harbor Laboratory
Date: 13-09-2021
DOI: 10.1101/2021.09.13.459777
Abstract: The ongoing SARS-CoV-2 pandemic has demonstrated the utility of real-time analysis of sequencing data, with a wide range of databases and resources for analysis now available. Here we show how the real-time nature of Oxford Nanopore Technologies sequencers can accelerate consensus generation, lineage and variant status assignment. We exploit the fact that multiplexed viral sequencing libraries quickly generate sufficient data for the majority of s les, with diminishing returns on remaining s les as the sequencing run progresses. We demonstrate methods to determine when a sequencing run has passed this point in order to reduce the time required and cost of sequencing. We extended MinoTour, our real-time analysis and monitoring platform for nanopore sequencers, to provide SARS-CoV2 analysis using ARTIC network pipelines. We additionally developed an algorithm to predict which s les will achieve sufficient coverage, automatically running the ARTIC medaka informatics pipeline once specific coverage thresholds have been reached on these s les. After testing on run data, we find significant run time savings are possible, enabling flow cells to be used more efficiently and enabling higher throughput data analysis. The resultant consensus genomes are assigned both PANGO lineage and variant status as defined by Public Health England. S les from within in idual runs are used to generate phylogenetic trees incorporating optional background s les as well as summaries of in idual SNPs. As minoTour uses ARTIC pipelines, new primer schemes and pathogens can be added to allow minoTour to aid in real-time analysis of pathogens in the future. Source code and documentation is available at github.com/LooseLab/minotourapp . Supplementary data are available from github.com/LooseLab/artic_minotour_analyses .
Publisher: American Society for Microbiology
Date: 10-2010
DOI: 10.1128/JB.00491-10
Abstract: Transcription of spa , encoding the virulence factor protein A in Staphylococcus aureus , is tightly controlled by a complex regulatory network, ensuring its temporal expression over growth and at appropriate stages of the infection process. Transcriptomic profiling of XdrA, a DNA-binding protein that is conserved in all S. aureus genomes and shares similarity with the XRE family of helix-turn-helix, antitoxin-like proteins, revealed it to be a previously unidentified activator of spa transcription. To assess how XdrA fits into the complex web of spa regulation, a series of regulatory mutants were constructed consisting of single, double, triple, and quadruple mutants lacking XdrA and/or the three key regulators previously shown to influence spa transcription directly (SarS, SarA, and RNAIII). A series of lacZ reporter gene fusions containing nested deletions of the spa promoter identified regions influenced by XdrA and the other three regulators. XdrA had almost as strong an activating effect on spa as SarS and acted on the same spa operator regions as SarS, or closely overlapping regions. All data from microarrays, Northern and Western blot analyses, and reporter gene fusion experiments indicated that XdrA is a major activator of spa expression that appears to act directly on the spa promoter and not through previously characterized regulators.
Publisher: Oxford University Press (OUP)
Date: 18-06-2012
DOI: 10.1111/J.1574-6968.2012.02603.X
Abstract: The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell ision. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.
Publisher: Cold Spring Harbor Laboratory
Date: 09-11-2017
DOI: 10.1101/211466
Abstract: Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus free or to have only exogenous variants of KoRV with low rates of KoRV induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV induced disease such as lymphoma. This paper uses a combination of sequencing technologies, Illumina RNA sequencing of “southern” (south Australian) and “northern” (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of “southern” (South Australian and Victorian animals) to retrieve full length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication competent KoRV, are not in fact KoRV free but harbour defective, presumably endogenous, “RecKoRV” variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV induced disease.
Publisher: Springer Science and Business Media LLC
Date: 27-01-2009
Abstract: Methicillin resistance in Staphylococcus aureus is conferred by the mecA -encoded penicillin-binding protein PBP2a. Additional genomic factors are also known to influence resistance levels in strain specific ways, although little is known about their contribution to resistance phenotypes in clinical isolates. Here we searched for novel proteins binding to the mec operator, in an attempt to identify new factor(s) controlling methicillin resistance phenotypes. Analysis of proteins binding to a DNA fragment containing the mec operator region identified a novel, putative helix-turn-helix DNA-binding protein, SA1665. Nonpolar deletion of SA1665, in heterogeneously methicillin resistant S. aureus (MRSA) of different genetic backgrounds, increased methicillin resistance levels in a strain dependent manner. This phenotype could be fully complemented by reintroducing SA1665 in trans. Northern and Western blot analyses, however, revealed that SA1665 had no visible influence on mecA transcription or amounts of PBP2a produced. SA1665 is a new chromosomal factor which influences methicillin resistance in MRSA. Although SA1665 bound to the mecA promoter region, it had no apparent influence on mecA transcription or translation, suggesting that this predicted DNA-binding protein modulates resistance indirectly, most likely through the control of other genomic factors which contribute to resistance.
Publisher: American Society for Microbiology
Date: 07-2006
DOI: 10.1128/AAC.00073-06
Abstract: Glycopeptide resistance, in a set of in vitro step-selected teicoplanin-resistant mutants derived from susceptible Staphylococcus aureus SA113, was associated with slower growth, thickening of the bacterial cell wall, increased N -acetylglucosamine incorporation, and decreased hemolysis. Differential transcriptome analysis showed that as resistance increased, some virulence-associated genes became downregulated. In a mouse tissue cage infection model, an inoculum of 10 4 CFU of strain SA113 rapidly produced a high-bacterial-load infection, which triggered MIP-2 release, leukocyte infiltration, and reduced leukocyte viability. In contrast, with the same inoculum of the isogenic glycopeptide-resistant derivative NM67, CFU initially decreased, resulting in the elimination of the mutant in three out of seven cages. In the four cages in which NM67 survived, it partially regained wild-type characteristics, including thinning of the cell wall, reduced N -acetylglucosamine uptake, and increased hemolysis however, the survivors also became teicoplanin hypersusceptible. The elimination of the teicoplanin-resistant mutants and selection of teicoplanin-hypersusceptible survivors in the tissue cages indicated that glycopeptide resistance imposes a fitness burden on S. aureus and is selected against in vivo, with restoration of fitness incurring the price of resistance loss.
Publisher: Cold Spring Harbor Laboratory
Date: 03-05-2018
DOI: 10.1101/312256
Abstract: The Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of s le types with erse methods of s le extraction. Nanopore sequencers output fast5 files containing signal data subsequently base called to fastq format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk fast5 file enabling inspection of signal in any channel at any point. We sought to visualise this signal to inspect challenging or difficult to sequence s les, or where flow cell performance is modified by an external agent, such as ‘Read Until’. The BulkVis tool can load a bulk fast5 file and overlays MinKNOW classifications on the signal trace. Users can navigate to a channel and time or, given a fastq header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly ided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2,272,580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input s le type and is more common in ‘ultra long’ read preparations. The software is available freely under an MIT license at github.com/LooseLab/bulkVis . The software requires python3 to run.
Publisher: American Society for Microbiology
Date: 08-2010
DOI: 10.1128/JCM.02113-09
Abstract: A cefoxitin-susceptible Staphylococcus aureus strain was identified by the Cepheid GeneXpert as methicillin-resistant S. aureus (MRSA). This strain was highly unstable and rapidly lost SCC mec upon subculturing in vitro , indicating that unstable MRSA is best detected by gene lification-based methods.
Publisher: Public Library of Science (PLoS)
Date: 03-03-2016
Publisher: American Society for Microbiology
Date: 11-2015
DOI: 10.1128/JCM.00202-15
Abstract: The control of food-borne outbreaks caused by Listeria monocytogenes in humans relies on the timely identification of food or environmental sources and the differentiation of outbreak-related isolates from unrelated ones. This study illustrates the utility of whole-genome sequencing for examining the link between clinical and environmental isolates of L. monocytogenes associated with an outbreak of hospital-acquired listeriosis in Sydney, Australia. Comparative genomic analysis confirmed an epidemiological link between the three clinical and two environmental isolates. Single nucleotide polymorphism (SNP) analysis showed that only two SNPs separated the three human outbreak isolates, which differed by 19 to 20 SNPs from the environmental isolates and 71 to ,000 SNPs from sporadic L. monocytogenes isolates. The chromosomes of all human outbreak isolates and the two suspected environmental isolates were syntenic. In contrast to the genomes of background sporadic isolates, all epidemiologically linked isolates contained two novel prophages and a previously unreported clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) locus subtype sequence. The mobile genetic element (MGE) profile of these isolates was distinct from that of the other serotype 1/2b reference strains and sporadic isolates. The identification of SNPs and clonally distinctive MGEs strengthened evidence to distinguish outbreak-related isolates of L. monocytogenes from cocirculating endemic strains.
Publisher: American Society for Microbiology
Date: 10-2005
DOI: 10.1128/JCM.43.10.5164-5170.2005
Abstract: The majority of methicillin-resistant Staphylococcus aureus (MRSA) isolates, recovered in 2003 at the Department of Medical Microbiology in Zürich, Switzerland, belonged to major clones that are circulating worldwide. Staphylococcal cassette chromosome mec type IV (SCC mec -IV), harbored by half of the isolates, was found in sequence type 217 (ST217), which is an allelic variant of epidemic MRSA-15 (designated EMRSA-15), in a new local ST617 descending from clonal complex CC8 and in low-level oxacillin-resistant strains of multiple genetic lineages characteristic of community-onset MRSA. SCC mec -I, SCC mec- II, and SCC mec -III were in the minority, and four MRSA isolates had complex, rearranged SCC mec elements. A novel SCC mec -N1 of approximately 30 kb, associated with a dfrA gene and a ccr4 -related recombinase complex, was identified in a large number of low-level oxacillin-resistant isolates, which descended from the successful clonal complex CC45 and are spreading among intraveneous drug users. In contrast, the SCC mec types of oxacillin-resistant coagulase-negative staphylococci (MRCNS) were of completely different composition. SCC mec type I (SCC mec -I) and SCC mec -II were more frequent than in the MRSA, while fewer contained SCC mec -IV. The other MRCNS displayed 11 different, complex patterns, suggesting frequent recombination between different SCC mec elements. With one ccr -negative exception, these strains lified between one and three different ccr products, indicating either new varied complexes or multiple ccr loci. This suggests the presence of novel SCC mec types in MRCNS and no extensive interspecies SCC mec transfer between MRSA and MRCNS.
Publisher: Oxford University Press (OUP)
Date: 23-05-2011
DOI: 10.1111/J.1574-6968.2011.02303.X
Abstract: Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance SA0908 protected cells from autolysis and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell ision in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.
Publisher: Cold Spring Harbor Laboratory
Date: 03-02-2020
DOI: 10.1101/2020.02.03.926956
Abstract: Nanopore sequencers enable selective sequencing of single molecules in real time by in idually reversing the voltage across specific nanopores. Thus DNA molecules can be rejected and replaced with new molecules enabling targeted sequencing to enrich, deplete or achieve specific coverage in a set of reads to address a biological question. We previously demonstrated this method worked using dynamic time warping mapping signal to reference, but required significant compute and did not scale to gigabase references. Using direct base calling with GPU we can now scale to gigabase references. We enrich for specific chromosomes mapping against the human genome and we develop pipelines enriching low abundance organisms from mixed populations without prior knowledge of s le composition. Finally, we enrich panels including 25,600 exon targets from 10,000 human genes and 717 genes implicated in cancer. Using this approach we identify PML-RARA fusions in the NB4 cell line in under 15 hours sequencing. These methods can be used to efficiently screen any target panel of genes without specialised s le preparation using a single computer and suitably powerful GPU.
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.BBAGEN.2006.06.008
Abstract: The vancomycin stress induced transcriptome of the methicillin susceptible Staphylococcus aureus (MSSA) strain Newman was determined by microarray analysis. Subsets of the induced ORFs corresponded to those previously reported to be induced by vancomycin in the methicillin resistant S. aureus (MRSA) strains N315 and JH1, and/or by other cell wall active antibiotics in RN450 while other ORFs appeared to be induced strain specifically in Newman. Northern analyses showed that the induction pathway for several of the ORFs appeared to be altered in a number of clinical NARSA isolates. Induction was found to be dependent on inhibitory concentrations of antibiotics.
Publisher: Elsevier BV
Date: 04-2015
Publisher: Springer Science and Business Media LLC
Date: 23-08-2014
Publisher: European Centre for Disease Control and Prevention (ECDC)
Date: 13-08-2020
DOI: 10.2807/1560-7917.ES.2020.25.32.2001410
Abstract: We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.
Publisher: Cold Spring Harbor Laboratory
Date: 12-2021
DOI: 10.1101/2021.12.01.470722
Abstract: Adaptive s ling enables selection of in idual DNA molecules from sequencing libraries, a unique property of nanopore sequencing. Here we develop our adaptive s ling tool readfish to become “barcode-aware” enabling selection of different targets within barcoded s les or filtering out in idual barcodes. We show that multiple human genomes can be assessed for copy number and structural variation on a single sequencing flow cell using s le specific customised target panels on both GridION and PromethION devices.
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.IJMM.2008.01.015
Abstract: The reason for the extremely low-level oxacillin resistance in a so-called 'drug clone', a methicillin-resistant Staphylococcus aureus circulating among injection drug users in Zurich, Switzerland, could be traced back to the mecA promoter sequence and particularly to the strain's genetic background. Sequencing of its mec complex identified a point mutation (TATACT to TATATT), creating a perfect palindrome in the -10 region of the mecA promoter/operator region containing the binding sites for the mecA repressors MecI and BlaI. Two strains with vastly different beta-lactam resistance phenotypes, the low-level resistant drug clone type strain CHE482 and the highly homogeneously resistant strain COLn, were cured of their SCCmec elements and subsequently transformed with plasmids containing mecA under the control of either the wild-type or mutant promoter. Expression studies showed that this mutation had significant effects on both mecA transcription and corresponding PBP2a production, but only small effects on beta-lactam resistance levels within a given genetic background. A further mutation in the mecA ribosomal binding site (GGAGG to GGAGT), common to SCCmec type IV strains, was found to have no discernable effect on mecA transcription and PBP2a content, and only minimal effects on beta-lactam resistance. Factors associated with the genetic backgrounds into which these differently controlled mecA genes were introduced had a much higher impact on beta-lactam resistance levels than the rates of mecA transcription. The tight repression of mecA expression in this drug clone in the absence of beta-lactams could contribute to the apparent fitness of this fast growing strain.
Publisher: Springer Science and Business Media LLC
Date: 09-12-2019
DOI: 10.1038/S41592-019-0697-Z
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: Research Square Platform LLC
Date: 13-08-2019
Abstract: High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe methods for extracting intact RNA, poly-A selection, cDNA conversion for a portion of s le, and library preparation for both direct RNA and cDNA libraries.
Publisher: American Society for Microbiology
Date: 12-2004
DOI: 10.1128/AAC.48.12.4800-4807.2004
Abstract: An association between moenomycin resistance and vancomycin intermediate resistance in Staphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn 551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn 551 insertions in the fmtC ( mprF ) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn 551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as β-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.
Publisher: Cold Spring Harbor Laboratory
Date: 12-09-2021
DOI: 10.1101/2021.09.10.459783
Abstract: MinoTour offers a LIMS system for Oxford Nanopore Technology (ONT) sequencers, with real-time metrics and analysis available permanently for review. Integration of unique real-time automated analysis can reduce the time required to answer biological questions, including mapping and classification of sequence whilst a run is in progress. Real-time sequence data requires new methods of analysis which do not wait for the completion of a run and MinoTour provides a framework to allow users to exploit these features. Source code and documentation are available at github.com/LooseLab/minotourcli and github.com/LooseLab/minotourapp . Docker images are available from /adoni5/ , and can be installed using a preconfigured docker-compose script at github.com/LooseLab/minotour-docker . An ex le server is available at 137.44.59.170 .
Publisher: Cold Spring Harbor Laboratory
Date: 09-11-2018
DOI: 10.1101/459529
Abstract: High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology. MA holds shares in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. REW, WT, TG, JRT, JQ, NJL, JTS, NS, AB, MA, HEO, MJ, and ML received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. NL has received an honorarium to speak at an ONT company meeting. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. JTS, ML and MA received research funding from ONT.
Publisher: Microbiology Society
Date: 28-06-2022
DOI: 10.1099/JGV.0.001749
Abstract: Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of ‘southern’ (south Australian) and ‘northern’ (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of ‘southern’ (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, ‘RecKoRV’ variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.
Publisher: Oxford University Press (OUP)
Date: 20-11-2018
DOI: 10.1093/BIOINFORMATICS/BTY841
Abstract: The Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of s le types with erse methods of s le extraction. Nanopore sequencers output FAST5 files containing signal data subsequently base called to FASTQ format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk FAST5 file enabling inspection of signal in any channel at any point. We sought to visualize this signal to inspect challenging or difficult to sequence s les. The BulkVis tool can load a bulk FAST5 file and overlays MinKNOW (the software that controls ONT sequencers) classifications on the signal trace and can show mappings to a reference. Users can navigate to a channel and time or, given a FASTQ header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly ided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2 272 580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input s le type and is more common in ’ultra-long’ read preparations. The software is available freely under an MIT license at github.com/LooseLab/bulkvis. Supplementary data are available at Bioinformatics online.
Publisher: Oxford University Press (OUP)
Date: 06-2009
DOI: 10.1111/J.1574-6968.2009.01603.X
Abstract: MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.
Publisher: Cold Spring Harbor Laboratory
Date: 24-08-2020
DOI: 10.1101/2020.08.22.20176834
Abstract: COVID-19 continues to cause a pandemic, having infected more than 20 million people globally. Successful elimination of the SARS-CoV-2 virus will require an effective vaccine. However, the immune correlates of infection are currently poorly understood. While neutralizing antibodies are believed to be essential for protection against infection, the contribution of the neutralizing antibody response to resolution of SARS-CoV-2 infection has not yet been defined. In this study the antibody responses to the SARS-CoV-2 spike protein and nucleocapsid proteins were investigated in a UK patient cohort, using optimised immunoassays and a retrovirus-based pseudotype entry assay. It was discovered that in severe COVID-19 infections an early antibody response to both antigens was associated with improved prognosis of infection. While not all SARS-CoV-2-reactive sera were found to possess neutralizing antibodies, neutralizing potency of sera was found to be greater in patients who went on to resolve infection, compared with those that died from COVID-19. Furthermore, viral genetic variation in spike protein was found to influence the production of neutralizing antibodies. Infection with the recently described spike protein variant 614G produced higher levels of neutralizing antibodies when compared to viruses possessing the 614D variant. These findings support the assertion that vaccines targeting generation of neutralizing antibodies may be useful at limiting SARS-CoV-2 infection. Assessment of the antibody responses to SARS-CoV-2 at time of diagnosis will be a useful addition to the diagnostic toolkit, enabling stratification of clinical intervention for severe COVID-19 disease.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Nadine Holmes.