ABSTRACT
The use of k-mers to capture genetic variation in bacterial genome-wide association studies (bGWAS) has demonstrated its effectiveness in overcoming the plasticity of bacterial genomes by providing a comprehensive array of genetic variants in a genome set that is not confined to a single reference genome. However, little attempt has been made to interpret k-mers in the context of genome rearrangements, partly due to challenges in the exhaustive and high-throughput identification of genome structure and individual rearrangement events. Here, we present GWarrange, a pre- and post-bGWAS processing methodology that leverages the unique properties of k-mers to facilitate bGWAS for genome rearrangements. Repeat sequences are common instigators of genome rearrangements through intragenomic homologous recombination, and they are commonly found at rearrangement boundaries. Using whole-genome sequences, repeat sequences are replaced by short placeholder sequences, allowing the regions flanking repeats to be incorporated into relatively short k-mers. Then, locations of flanking regions in significant k-mers are mapped back to complete genome sequences to visualise genome rearrangements. Four case studies based on two bacterial species (Bordetella pertussis and Enterococcus faecium) and a simulated genome set are presented to demonstrate the ability to identify phenotype-associated rearrangements. GWarrange is available at https://github.com/DorothyTamYiLing/GWarrange.
Subject(s)
Gene Rearrangement , Genome, Bacterial , Genome-Wide Association Study , Phenotype , Genome-Wide Association Study/methods , Software , Genetic VariationABSTRACT
Accurate annotation to single-nucleotide resolution of the transcribed regions in genomes is key to optimally analyse RNA-seq data, understand regulatory events and for the design of experiments. However, currently most genome annotations provided by GenBank generally lack information about untranslated regions. Additionally, information regarding genomic locations of non-coding RNAs, such as sRNAs, or anti-sense RNAs is frequently missing. To provide such information, diverse RNA-seq technologies, such as Rend-seq, have been developed and applied to many bacterial species. However, incorporating this vast amount of information into annotation files has been limited and is bioinformatically challenging, resulting in UTRs and other non-coding elements being overlooked or misrepresented. To overcome this problem, we present pyRAP (python Rend-seq Annotation Pipeline), a software package that analyses Rend-seq datasets to accurately resolve transcript boundaries genome-wide. We report the use of pyRAP to find novel transcripts, transcript isoforms, and RNase-dependent sRNA processing events. In Bacillus subtilis we uncovered 63 novel transcripts and provide genomic coordinates with single-nucleotide resolution for 2218 5'UTRs, 1864 3'UTRs and 161 non-coding RNAs. In Escherichia coli, we report 117 novel transcripts, 2429 5'UTRs, 1619 3'UTRs and 91 non-coding RNAs, and in Staphylococcus aureus, 16 novel transcripts, 664 5'UTRs, 696 3'UTRs, and 81 non-coding RNAs. Finally, we use pyRAP to produce updated annotation files for B. subtilis 168, E. coli K-12 MG1655, and S. aureus 8325 for use in the wider microbial genomics research community.
Subject(s)
Bacillus subtilis , Genome, Bacterial , Molecular Sequence Annotation , Software , Bacillus subtilis/genetics , Escherichia coli/genetics , RNA, Bacterial/genetics , Staphylococcus aureus/genetics , Computational Biology/methods , Sequence Analysis, RNA/methods , RNA-Seq/methodsABSTRACT
Salmonella enterica serovar Enteritidis is one of the most frequent causes of Salmonellosis globally and is commonly transmitted from animals to humans by the consumption of contaminated foodstuffs. In the UK and many other countries in the Global North, a significant proportion of cases are caused by the consumption of imported food products or contracted during foreign travel, therefore, making the rapid identification of the geographical source of new infections a requirement for robust public health outbreak investigations. Herein, we detail the development and application of a hierarchical machine learning model to rapidly identify and trace the geographical source of S. Enteritidis infections from whole genome sequencing data. 2313 S. Enteritidis genomes, collected by the UKHSA between 2014-2019, were used to train a 'local classifier per node' hierarchical classifier to attribute isolates to four continents, 11 sub-regions, and 38 countries (53 classes). The highest classification accuracy was achieved at the continental level followed by the sub-regional and country levels (macro F1: 0.954, 0.718, 0.661, respectively). A number of countries commonly visited by UK travelers were predicted with high accuracy (hF1: >0.9). Longitudinal analysis and validation with publicly accessible international samples indicated that predictions were robust to prospective external datasets. The hierarchical machine learning framework provided granular geographical source prediction directly from sequencing reads in <4 min per sample, facilitating rapid outbreak resolution and real-time genomic epidemiology. The results suggest additional application to a broader range of pathogens and other geographically structured problems, such as antimicrobial resistance prediction, is warranted.
Subject(s)
Salmonella Infections , Salmonella enterica , Animals , Humans , Salmonella enteritidis/genetics , Prospective Studies , Salmonella Infections/epidemiology , Disease Outbreaks , Machine LearningABSTRACT
Shigella sonnei causes shigellosis, a severe gastrointestinal illness that is sexually transmissible among men who have sex with men (MSM). Multidrug resistance in S. sonnei is common including against World Health Organisation recommended treatment options, azithromycin, and ciprofloxacin. Recently, an MSM-associated outbreak of extended-spectrum ß-lactamase producing, extensively drug resistant S. sonnei was reported in the United Kingdom. Here, we aimed to identify the genetic basis, evolutionary history, and international dissemination of the outbreak strain. Our genomic epidemiological analyses of 3,304 isolates from the United Kingdom, Australia, Belgium, France, and the United States of America revealed an internationally connected outbreak with a most recent common ancestor in 2018 carrying a low-fitness cost resistance plasmid, previously observed in travel associated sublineages of S. flexneri. Our results highlight the persistent threat of horizontally transmitted antimicrobial resistance and the value of continuing to work towards early and open international sharing of genomic surveillance data.
Subject(s)
Sexual and Gender Minorities , Shigella , Male , Humans , Shigella sonnei/genetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Homosexuality, Male , Travel , Drug Resistance, Bacterial/genetics , Microbial Sensitivity TestsABSTRACT
BACKGROUND: Young adults affected by cancer face physical and psychological challenges and desire online supportive care. Yoga can be delivered online and may improve physical and psychological outcomes. Yet, yoga has rarely been studied with young adults affected by cancer. To address this, an 8-week yoga intervention was developed, and a pilot study was deemed necessary to explore feasibility, acceptability, implementation, and potential benefits. METHODS: A mixed-methods, single-arm hybrid effectiveness-implementation pilot study evaluating the yoga intervention was conducted. Feasibility was assessed by tracking enrollment, retention, attendance, completeness of data, and adverse events. Acceptability was explored through interviews. Implementation metrics included training time, delivery resources, and fidelity. Potential effectiveness was evaluated by exploring changes in physical (i.e., balance, flexibility, range of motion, functional mobility) and psychological (i.e., quality of life, fatigue, resilience, posttraumatic growth, body image, mindfulness, perceived stress) outcomes at pre- (week 0), post- (week 8), and follow-up (week 16) time points. Data were analyzed with descriptive statistics, repeated measures analysis of variance, and content analysis. RESULTS: Thirty young adults participated in this study (recruitment rate = 33%). Retention to study procedures was 70%, and attendance ranged from 38 to 100%. There were little missing data (< 5%) and no adverse events. Though most participants were satisfied with the yoga intervention, recommendations for improvement were shared. Sixty study-specific training hours and > 240 delivery and assessment hours were accrued and fidelity was high. Functional mobility, flexibility, quality of life (energy/fatigue, social well-being), body image (appearance evaluation), mindfulness (non-reactivity), and perceived stress improved significantly over time (all p< 0.050; [Formula: see text]). No other significant changes were observed (all p> 0.050; [Formula: see text]). CONCLUSIONS: The yoga intervention may confer physical and psychological benefits, though intervention and study-specific modifications are required to improve feasibility and acceptability. Requiring study participation and providing greater scheduling flexibility could enhance recruitment and retention. Increasing the frequency of classes offered each week and offering more opportunities for participant interaction could improve satisfaction. This study highlights the value of doing pilot work and provides data that has directly informed intervention and study modifications. Findings could also be used by others offering yoga or supportive care by videoconference to young adults affected by cancer. TRIAL REGISTRATION: Not available-not registered.
ABSTRACT
Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to ß-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.
Subject(s)
Amoxicillin , Streptococcus pneumoniae , Amoxicillin/metabolism , Amoxicillin/pharmacology , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Transfer, Horizontal , Humans , Microbial Sensitivity Tests , Penicillin Resistance/genetics , Penicillin-Binding Proteins/genetics , Streptococcus pneumoniae/metabolismABSTRACT
Reviewing the genetics underlying the arms race between bacteria and bacteriophages can offer an interesting insight into the development of bacterial resistance and phage co-evolution. This study shows how the natural development of resistances to the K1F bacteriophage, a phage which targets the K1 capsule of pathogenic Escherichia coli, can come about through insertion sequences (IS). Of the K1F resistant mutants isolated, two were of particular interest. The first of these showed full resistance to K1F and was found to have disruptions to kpsE, the product of which is involved in polysialic acid translocation. The second, after showing an initial susceptibility to K1F which then developed to full resistance, had disruptions to neuC, a gene involved in one of the early steps of polysialic acid biosynthesis. Both of these mutations came with a fitness cost and produced considerable phenotypic differences in the completeness and location of the K1 capsule when compared with the wild type. Sequential treatment of these two K1F resistant mutants with T7 resulted in the production of a variety of isolates, many of which showed a renewed susceptibility to K1F, indicating that these insertion sequence mutations are reversible, as well as one isolate that developed resistance to both phages. IMPORTANCE Bacteriophages have many potential uses in industry and the clinical environment as an antibacterial control measure. One of their uses, phage therapy, is an appealing alternative to antibiotics due to their high specificity. However, as with the rise in antimicrobial resistance (AMR), it is critical to improve our understanding of how resistance develops against these viral agents. In the same way as bacteria will evolve and mutate antibiotic receptors so they can no longer be recognized, resistance to bacteriophages can come about via mutations to phage receptors, preventing phage binding and infection. We have shown that Escherichia coli will become resistant to the K1F bacteriophage via insertion element reshufflings causing null mutations to elements of the polysialic acid biosynthetic cluster. Exposure to the T7 bacteriophage then resulted in further changes in the position of these IS elements, further altering their resistance and sensitivity profiles.
Subject(s)
Bacteriophages , Escherichia coli Proteins , Escherichia coli , Bacteriophages/genetics , DNA Transposable Elements , Escherichia coli/genetics , Escherichia coli/virology , Escherichia coli Proteins/genetics , Membrane Transport Proteins/genetics , Multigene Family , Sialic AcidsABSTRACT
Genomics, combined with population mobility data, used to map importation and spatial spread of SARS-CoV-2 in high-income countries has enabled the implementation of local control measures. Here, to track the spread of SARS-CoV-2 lineages in Bangladesh at the national level, we analysed outbreak trajectory and variant emergence using genomics, Facebook 'Data for Good' and data from three mobile phone operators. We sequenced the complete genomes of 67 SARS-CoV-2 samples (collected by the IEDCR in Bangladesh between March and July 2020) and combined these data with 324 publicly available Global Initiative on Sharing All Influenza Data (GISAID) SARS-CoV-2 genomes from Bangladesh at that time. We found that most (85%) of the sequenced isolates were Pango lineage B.1.1.25 (58%), B.1.1 (19%) or B.1.36 (8%) in early-mid 2020. Bayesian time-scaled phylogenetic analysis predicted that SARS-CoV-2 first emerged during mid-February in Bangladesh, from abroad, with the first case of coronavirus disease 2019 (COVID-19) reported on 8 March 2020. At the end of March 2020, three discrete lineages expanded and spread clonally across Bangladesh. The shifting pattern of viral diversity in Bangladesh, combined with the mobility data, revealed that the mass migration of people from cities to rural areas at the end of March, followed by frequent travel between Dhaka (the capital of Bangladesh) and the rest of the country, disseminated three dominant viral lineages. Further analysis of an additional 85 genomes (November 2020 to April 2021) found that importation of variant of concern Beta (B.1.351) had occurred and that Beta had become dominant in Dhaka. Our interpretation that population mobility out of Dhaka, and travel from urban hotspots to rural areas, disseminated lineages in Bangladesh in the first wave continues to inform government policies to control national case numbers by limiting within-country travel.
Subject(s)
COVID-19/transmission , Cell Phone/statistics & numerical data , Genome, Viral/genetics , SARS-CoV-2/genetics , Social Media/statistics & numerical data , Bangladesh/epidemiology , Bayes Theorem , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Disease Outbreaks/prevention & control , Disease Outbreaks/statistics & numerical data , Genomics , Health Policy/legislation & jurisprudence , Humans , Phylogeny , Population Dynamics/statistics & numerical data , SARS-CoV-2/classification , Travel/legislation & jurisprudence , Travel/statistics & numerical dataABSTRACT
Shigellosis in men who have sex with men (MSM) is caused by multidrug resistant Shigellae, exhibiting resistance to antimicrobials including azithromycin, ciprofloxacin and more recently the third-generation cephalosporins. We sequenced four blaCTX-M-27-positive MSM Shigella isolates (2018-20) using Oxford Nanopore Technologies; three S. sonnei (identified as two MSM clade 2, one MSM clade 5) and one S. flexneri 3a, to explore AMR context. All S. sonnei isolates harboured Tn7/Int2 chromosomal integrons, whereas S. flexneri 3a contained the Shigella Resistance Locus. All strains harboured IncFII pKSR100-like plasmids (67-83kbp); where present blaCTX-M-27 was located on these plasmids flanked by IS26 and IS903B, however blaCTX-M-27 was lost in S. flexneri 3a during storage between Illumina and Nanopore sequencing. IncFII AMR regions were mosaic and likely reorganised by IS26; three of the four plasmids contained azithromycin-resistance genes erm(B) and mph(A) and one harboured the pKSR100 integron. Additionally, all S. sonnei isolates possessed a large IncB/O/K/Z plasmid, two of which carried aph(3')-Ib/aph(6)-Id/sul2 and tet(A). Monitoring the transmission of mobile genetic elements with co-located AMR determinants is necessary to inform empirical treatment guidance and clinical management of MSM-associated shigellosis.
Subject(s)
Bacterial Proteins/genetics , Dysentery, Bacillary/transmission , Homosexuality, Male , Plasmids/genetics , Sexual and Gender Minorities , Shigella/genetics , beta-Lactamases/genetics , Adult , Anti-Bacterial Agents/therapeutic use , DNA, Bacterial , Dysentery, Bacillary/drug therapy , Humans , Male , Middle Aged , Nanopores , Shigella/classification , Shigella sonnei/genetics , Shigella sonnei/isolation & purification , United Kingdom , Virulence/genetics , Young AdultABSTRACT
Surveillance of drug-resistant bacteria is essential for healthcare providers to deliver effective empirical antibiotic therapy. However, traditional molecular epidemiology does not typically occur on a timescale that could affect patient treatment and outcomes. Here, we present a method called 'genomic neighbour typing' for inferring the phenotype of a bacterial sample by identifying its closest relatives in a database of genomes with metadata. We show that this technique can infer antibiotic susceptibility and resistance for both Streptococcus pneumoniae and Neisseria gonorrhoeae. We implemented this with rapid k-mer matching, which, when used on Oxford Nanopore MinION data, can run in real time. This resulted in the determination of resistance within 10 min (91% sensitivity and 100% specificity for S. pneumoniae and 81% sensitivity and 100% specificity for N. gonorrhoeae from isolates with a representative database) of starting sequencing, and within 4 h of sample collection (75% sensitivity and 100% specificity for S. pneumoniae) for clinical metagenomic sputum samples. This flexible approach has wide application for pathogen surveillance and may be used to greatly accelerate appropriate empirical antibiotic treatment.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Typing Techniques/methods , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Genomics , Databases, Factual , Humans , Microbial Sensitivity Tests/methods , Molecular Epidemiology , Neisseria gonorrhoeae/drug effects , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/isolation & purification , Phenotype , Sensitivity and Specificity , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/isolation & purificationABSTRACT
Bacterial-host interactions are non-linear and actually threefold, involving significant selection through predatory lytic bacteriophages in the host environment. In studies of human and animal gut microbiome bacteria, it is important to consider phage in all host-pathogen interactions. We use an important zoonotic pathogen, Shiga toxigenic Escherichia coli (STEC) O157:H7, to investigate this. Our study provides evidence that phage resistance profiles are well maintained at the sub-lineage level with variation in profiles within sub-lineages uncommon. This indicates that phage resistance heterogeneity happened early on in the STEC O157:H7 natural history and that occasional "wobbles" do not often outcompete the stable lineage unless combined with a competitive advantage. We discuss an example of this in the acquisition of stx2a that, while an important virulence factor, also conveys increased phage cross-resistance. We also discuss the role of phage resistance in co-occurrence of the three stable lineages worldwide and whether differing phage resistance is maintaining diversity.
ABSTRACT
Temperate bacteriophages are a common feature of Pseudomonas aeruginosa genomes, but their role in chronic lung infections is poorly understood. This study was designed to identify the diverse communities of mobile P. aeruginosa phages by employing novel metagenomic methods, to determine cross infectivity, and to demonstrate the influence of phage infection on antimicrobial susceptibility. Mixed temperate phage populations were chemically mobilized from individual P. aeruginosa, isolated from patients with cystic fibrosis (CF) or bronchiectasis (BR). The infectivity phenotype of each temperate phage lysate was evaluated by performing a cross-infection screen against all bacterial isolates and tested for associations with clinical variables. We utilized metagenomic sequencing data generated for each phage lysate and developed a novel bioinformatic approach allowing resolution of individual temperate phage genomes. Finally, we used a subset of the temperate phages to infect P. aeruginosa PAO1 and tested the resulting lysogens for their susceptibility to antibiotics. Here, we resolved 105 temperate phage genomes from 94 lysates that phylogenetically clustered into 8 groups. We observed disease-specific phage infectivity profiles and found that phages induced from bacteria isolated from more advanced disease infected broader ranges of P. aeruginosa isolates. Importantly, when infecting PAO1 in vitro with 20 different phages, 8 influenced antimicrobial susceptibility. This study shows that P. aeruginosa isolated from CF and BR patients harbors diverse communities of inducible phages, with hierarchical infectivity profiles that relate to the progression of the disease. Temperate phage infection altered the antimicrobial susceptibility of PAO1 at subinhibitory concentrations of antibiotics, suggesting they may be precursory to antimicrobial resistance.IMPORTANCE Pseudomonas aeruginosa is a key opportunistic respiratory pathogen in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. The genomes of these pathogens are enriched with mobile genetic elements including diverse temperate phages. While the temperate phages of the Liverpool epidemic strain have been shown to be active in the human lung and enhance fitness in a rat lung infection model, little is known about their mobilization more broadly across P. aeruginosa in chronic respiratory infection. Using a novel metagenomic approach, we identified eight groups of temperate phages that were mobilized from 94 clinical P. aeruginosa isolates. Temperate phages from P. aeruginosa isolated from more advanced disease showed high infectivity rates across a wide range of P. aeruginosa genotypes. Furthermore, we showed that multiple phages altered the susceptibility of PAO1 to antibiotics at subinhibitory concentrations.
ABSTRACT
BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.
Subject(s)
Amides/therapeutic use , Antiviral Agents/therapeutic use , Ebolavirus/drug effects , Hemorrhagic Fever, Ebola/drug therapy , Pyrazines/therapeutic use , Adolescent , Adult , Child , Child, Preschool , Compassionate Use Trials/methods , Female , Guinea , Hemorrhagic Fever, Ebola/virology , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Retrospective Studies , Viral Load/drug effects , Young AdultABSTRACT
Whole genome sequencing in conjunction with traditional epidemiology has been used to reconstruct transmission networks of Mycobacterium tuberculosis during outbreaks. Given its low mutation rate, genetic diversity within M. tuberculosis outbreaks can be extremely limited - making it difficult to determine precisely who transmitted to whom. In addition to consensus SNPs (cSNPs), examining heterogeneous alleles (hSNPs) has been proposed to improve resolution. However, few studies have examined the potential biases in detecting these hSNPs. Here, we analysed genome sequence data from 25 specimens from British Columbia, Canada. Specimens were sequenced to a depth of 112-296×. We observed biases in read depth, base quality, strand distribution and read placement where possible hSNPs were initially identified, so we applied conservative filters to reduce false positives. Overall, there was phylogenetic concordance between the observed 2542 cSNP and 63 hSNP loci. Furthermore, we identified hSNPs shared exclusively by epidemiologically linked patients, supporting their use in transmission inferences. We conclude that hSNPs may add resolution to transmission networks, particularly where the overall genetic diversity is low.
Subject(s)
Disease Outbreaks , Genome, Bacterial , Mutation Rate , Mycobacterium tuberculosis/genetics , Phylogeny , Polymorphism, Single Nucleotide , Tuberculosis, Pulmonary , Whole Genome Sequencing , British Columbia/epidemiology , Humans , Mycobacterium tuberculosis/isolation & purification , Tuberculosis, Pulmonary/epidemiology , Tuberculosis, Pulmonary/genetics , Tuberculosis, Pulmonary/transmissionABSTRACT
Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.
Subject(s)
Bacteriophage T4/growth & development , Bacteriophage T7/growth & development , Escherichia coli O157/virology , Genes, Bacterial , Host-Parasite Interactions , DNA Transposable Elements , Escherichia coli O157/genetics , Mutagenesis, Insertional , Sequence Analysis, DNAABSTRACT
Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.
Subject(s)
Cell Communication/genetics , Recombination, Genetic/genetics , Streptococcus pneumoniae/genetics , Cell Communication/physiology , DNA/genetics , DNA/physiology , Evolution, Molecular , Gene Rearrangement/genetics , Genome, Bacterial/genetics , Homologous Recombination/genetics , Polymorphism, Single Nucleotide/genetics , Whole Genome Sequencing/methodsABSTRACT
We compared the genomes of 60 isolates of enteroinvasive Escherichia coli (EIEC) in order to better understand the step-wise evolutionary process from non-pathogenic to host-adapted pathogenic E. coli. All isolates belonged to either sequence type (ST) 6, ST99 or ST270. Each ST was located on different branches of the E. coli phylogeny and had invasion plasmids (pINVs) belonging to FII-21 (ST99, ST270), FII-27 (ST270) or FII-28 (ST6, ST270) incompatibility groups. A higher number of insertion sequence (IS) elements were identified in ST6 and ST270 than in ST99, and appeared to be driving the loss of functional genes. Comparison of the pINV from each ST revealed different degrees of gene loss, with pINV from ST270 being most similar to that found in Shigella species. We captured three EIEC STs at different stages of patho-adaptation, with ST270 being the most 'shigella-like' and the most divergent from non-pathogenic E. coli, and ST99 being the least divergent.
ABSTRACT
On 11 May 2015, the Dubréka prefecture, Guinea, reported nine laboratory-confirmed cases of Ebola virus disease (EVD). None could be epidemiologically linked to cases previously reported in the prefecture. We describe the epidemiological and molecular investigations of this event. We used the Dubréka EVD registers and the Ebola treatment centre's (ETC) records to characterise chains of transmission. Real-time field Ebola virus sequencing was employed to support epidemiological results. An epidemiological cluster of 32 cases was found, of which 27 were laboratory confirmed, 24 were isolated and 20 died. Real-time viral sequencing on 12 cases demonstrated SL3 lineage viruses with sequences differing by one to three nt inside a single phylogenetic cluster. For isolated cases, the average time between symptom onset and ETC referral was 2.8 days (interquartile range (IQR): 1-4). The average time between sample collection and molecular results' availability was 3 days (IQR: 2-5). In an area with scarce resources, the genetic characterisation supported the outbreak investigations in real time, linking cases where epidemiological investigation was limited and reassuring that the responsible strain was already circulating in Guinea. We recommend coupling thorough epidemiological and genomic investigations to control EVD clusters.
Subject(s)
DNA, Viral/genetics , Ebolavirus/genetics , Ebolavirus/isolation & purification , Hemorrhagic Fever, Ebola/diagnosis , Hemorrhagic Fever, Ebola/transmission , Contact Tracing , Disease Outbreaks/prevention & control , Genomics , Guinea/epidemiology , Hemorrhagic Fever, Ebola/epidemiology , Hemorrhagic Fever, Ebola/virology , Humans , Phylogeny , Real-Time Polymerase Chain ReactionABSTRACT
In July 2014, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a-encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern.