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Volume 8,
Issue 9,
2022
Volume 8, Issue 9, 2022
- Editorials
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- Research Articles
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- Genomic Methodologies
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Ribosomal MLST nucleotide identity (rMLST-NI), a rapid bacterial species identification method: application to Klebsiella and Raoultella genomic species validation
Bacterial genomics is making an increasing contribution to the fields of medicine and public health microbiology. Consequently, accurate species identification of bacterial genomes is an important task, particularly as the number of genomes stored in online databases increases rapidly and new species are frequently discovered. Existing database entries require regular re-evaluation to ensure that species annotations are consistent with the latest species definitions. We have developed an automated method for bacterial species identification that is an extension of ribosomal multilocus sequence typing (rMLST). The method calculates an ‘rMLST nucleotide identity’ (rMLST-NI) based on the nucleotides present in the protein-encoding ribosomal genes derived from bacterial genomes. rMLST-NI was used to validate the species annotations of 11839 publicly available Klebsiella and Raoultella genomes based on a comparison with a library of type strain genomes. rMLST-NI was compared with two whole-genome average nucleotide identity methods (OrthoANIu and FastANI) and the k-mer based Kleborate software. The results of the four methods agreed across a dataset of 11839 bacterial genomes and identified a small number of entries (n=89) with species annotations that required updating. The rMLST-NI method was 3.5 times faster than Kleborate, 4.5 times faster than FastANI and 1600 times faster than OrthoANIu. rMLST-NI represents a fast and generic method for species identification using type strains as a reference.
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In silico identification of bacterial seaweed-degrading bioplastic producers
More LessThere is an urgent need to replace petroleum-based plastic with bio-based and biodegradable alternatives. Polyhydroxyalkanoates (PHAs) are attractive prospective replacements that exhibit desirable mechanical properties and are recyclable and biodegradable in terrestrial and marine environments. However, the production costs today still limit the economic sustainability of the PHA industry. Seaweed cultivation represents an opportunity for carbon capture, while also supplying a sustainable photosynthetic feedstock for PHA production. We mined existing gene and protein databases to identify bacteria able to grow and produce PHAs using seaweed-derived carbohydrates as substrates. There were no significant relationships between the genes involved in the deconstruction of algae polysaccharides and PHA production, with poor to negative correlations and diffused clustering suggesting evolutionary compartmentalism. We identified 2 987 bacterial candidates spanning 40 taxonomic families predominantly within Alphaproteobacteria, Gammaproteobacteria and Burkholderiales with enriched seaweed-degrading capacity that also harbour PHA synthesis potential. These included highly promising candidates with specialist and generalist specificities, including Alteromonas, Aquisphaera, Azotobacter, Bacillus, Caulobacter, Cellvibrionaceae, Duganella, Janthinobacterium, Massilia, Oxalobacteraceae, Parvularcula, Pirellulaceae, Pseudomonas, Rhizobacter, Rhodanobacter, Simiduia, Sphingobium, Sphingomonadaceae, Sphingomonas, Stieleria, Vibrio and Xanthomonas. In this enriched subset, the family-level densities of genes targeting green macroalgae polysaccharides were considerably higher (n=231.6±68.5) than enzymes targeting brown (n=65.34±13.12) and red (n=30.5±10.72) polysaccharides. Within these organisms, an abundance of FabG genes was observed, suggesting that the fatty acid de novo synthesis pathway supplies (R)−3-hydroxyacyl-CoA or 3-hydroxybutyryl-CoA from core metabolic processes and is the predominant mechanism of PHA production in these organisms. Our results facilitate extending seaweed biomass valorization in the context of consolidated biorefining for the production of bioplastics.
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A general approach to identify low-frequency variants within influenza samples collected during routine surveillance
Influenza viruses exhibit considerable diversity between hosts. Additionally, different quasispecies can be found within the same host. High-throughput sequencing technologies can be used to sequence a patient-derived virus population at sufficient depths to identify low-frequency variants (LFV) present in a quasispecies, but many challenges remain for reliable LFV detection because of experimental errors introduced during sample preparation and sequencing. High genomic copy numbers and extensive sequencing depths are required to differentiate false positive from real LFV, especially at low allelic frequencies (AFs). This study proposes a general approach for identifying LFV in patient-derived samples obtained during routine surveillance. Firstly, validated thresholds were determined for LFV detection, whilst balancing both the cost and feasibility of reliable LFV detection in clinical samples. Using a genetically well-defined population of influenza A viruses, thresholds of at least 104 genomes per microlitre and AF of ≥5 % were established as detection limits. Secondly, a subset of 59 retained influenza A (H3N2) samples from the 2016–2017 Belgian influenza season was composed. Thirdly, as a proof of concept for the added value of LFV for routine influenza monitoring, potential associations between patient data and whole genome sequencing data were investigated. A significant association was found between a high prevalence of LFV and disease severity. This study provides a general methodology for influenza LFV detection, which can also be adopted by other national influenza reference centres and for other viruses such as SARS-CoV-2. Additionally, this study suggests that the current relevance of LFV for routine influenza surveillance programmes might be undervalued.
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Single-cell genomics reveals population structures from in vitro evolutionary studies of Salmonella
Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer but have not yet been deployed to study evolving clonal microbial populations. Here, we present an approach for single bacterial genomic analysis for in vitro evolution experiments using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to the experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from populations we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution.
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- Functional Genomics and Microbe–Niche Interactions
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Comparative genomics of Rothia species reveals diversity in novel biosynthetic gene clusters and ecological adaptation to different eukaryotic hosts and host niches
Rothia species are understudied members of the phylum Actinobacteria and prevalent colonizers of the human and animal upper respiratory tract and oral cavity. The oral cavity, including the palatine tonsils, is colonized by a complex microbial community, which compete for resources, actively suppress competitors and influence host physiology. We analysed genomic data from 43 new porcine Rothia isolates, together with 112 publicly available draft genome sequences of Rothia isolates from humans, animals and the environment. In all Rothia genomes, we identified biosynthetic gene clusters predicted to produce antibiotic non-ribosomal peptides, iron scavenging siderophores and other secondary metabolites that modulate microbe–microbe and potentially microbe–host interactions. In vitro overlay inhibition assays corroborated the hypothesis that specific strains produce natural antibiotics. Rothia genomes encode a large number of carbohydrate-active enzymes (CAZy), with varying CAZy activities among the species found in different hosts, host niches and environments. These findings reveal competition mechanisms and metabolic specializations linked to ecological adaptation of Rothia species in different hosts.
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The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses
More LessChemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.
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Phylogenomic analysis of the genus Delftia reveals distinct major lineages with ecological specializations
More LessDelftia is a diverse betaproteobacterial genus with many strains having agricultural and industrial relevance, including plant-growth promotion, bioremediation of hydrocarbon-contaminated soils, and heavy metal immobilization. Delftia spp. are broadly distributed in the environment, and have been isolated from plant hosts as well as healthy and diseased animal hosts, yet the genetic basis of this ecological versatility has not been characterized. Here, we present a phylogenomic comparison of published Delftia genomes and show that the genus is divided into two well-supported clades: one ‘ Delftia acidovorans ’ clade with isolates from soils and plant rhizospheres, and a second ‘ Delftia lacustris and Delftia tsuruhatensis ’ clade with isolates from humans and sludge. The pan-genome inferred from 61 Delftia genomes contained over 28 000 genes, of which only 884 were found in all genomes. Analysis of industrially relevant functions highlighted the ecological versatility of Delftia and supported their role as generalists.
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Genome analysis of a new sulphur disproportionating species Thermosulfurimonas strain F29 and comparative genomics of sulfur-disproportionating bacteria from marine hydrothermal vents
More LessThis paper reports on the genome analysis of strain F29 representing a new species of the genus Thermosulfurimonas . This strain, isolated from the Lucky Strike hydrothermal vent field on the Mid-Atlantic Ridge, is able to grow by disproportionation of S0 with CO2 as a carbon source. Strain F29 possesses a genome of 2,345,565 bp, with a G+C content of 58.09%, and at least one plasmid. The genome analysis revealed complete sets of genes for CO2 fixation via the Wood–Ljungdahl pathway, for sulphate-reduction and for hydrogen oxidation, suggesting the involvement of the strain into carbon, sulphur, and hydrogen cycles of deep-sea hydrothermal vents. Strain F29 genome encodes also several CRISPR sequences, suggesting that the strain may be subjected to viral attacks. Comparative genomics was carried out to decipher sulphur disproportionation pathways. Genomes of sulphur-disproportionating bacteria from marine hydrothermal vents were compared to the genomes of non-sulphur-disproportionating bacteria. This analysis revealed the ubiquitous presence in these genomes of a molybdopterin protein consisting of a large and a small subunit, and an associated chaperone. We hypothesize that these proteins may be involved in the process of elemental sulphur disproportionation.
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- Microbial Communities
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Changes to the upper gastrointestinal microbiotas of children with reflux oesophagitis and oesophageal metaplasia
Little is known of the relationships among paediatric upper gastrointestinal microbiotas, and the impact of medication use and disease on their diversity. Here, we investigated the diversity of three microbiotas in the upper gastrointestinal tract of paediatric patients in relation to each other and to host factors. Oral, oesophageal and gastric microbiotas from a prospective paediatric cohort (n=54) were profiled using the 16S rRNA gene and ITS2 amplicon sequencing. 16S rRNA gene amplicon sequencing of oesophageal biopsies from a retrospective paediatric cohort (n=96) and shotgun metagenomics data from oesophageal brushings (n=88) were employed for genomic signature validation. Bacterial diversity and composition showed substantial differences across oral, oesophageal and gastric fluid samples that were not replicated for fungi, and the presence of reflux led to increased homogeneity in the bacterial component of these three microbiotas. The oral and oesophageal microbiotas were associated with age, sex, history of oesophageal atresia and presence of oesophageal metaplasia, with the latter characterized by Prevotella enrichment. Proton pump inhibitor use was associated with increased oral bacterial richness in the gastric fluid, and this correlated with increased levels of gastric pro-inflammatory cytokines. Profiling of oesophageal biopsies from a retrospective paediatric cohort confirmed an increased Prevotella prevalence in samples with metaplasia. Analysis of metagenome-derived oesophageal Prevotella melaninogenica genomes identified strain-specific features that were significantly increased in prevalence in samples with metaplasia. Prevotella enrichment is a signature associated with paediatric oesophageal metaplasia, and proton pump inhibitor use substantially alters the paediatric gastric microenvironment.
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- Pathogens and Epidemiology
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Key features of pneumococcal isolates recovered in Central and Northwestern Russia in 2011–2018 determined through whole-genome sequencing
Invasive pneumococcal disease remains one of the leading causes of morbidity and mortality worldwide. In Russia, 13- valent pneumococcal conjugate vaccine (PCV13) was introduced into the childhood immunization programme nationwide in 2014. As part of the Global Pneumococcal Sequencing Project (GPS), we used genome data to characterize 179 pneumococcal isolates collected from Russia in 2011–2018 to investigate the circulating pneumococcal strains using a standardized genomic definition of pneumococcal lineages (global pneumococcal sequence clusters, GPSCs), prevalent serotypes and antimicrobial resistance profiles.
We observed high serotype and lineage diversity among the 179 isolates recovered from cerebrospinal fluid (n=77), nasopharyngeal swabs (n=99) and other non-sterile site swabs (n=3). Overall, 60 GPSCs were identified, including 48 clonal complexes (CCs) and 14 singletons, and expressed 42 serotypes (including non-typable). Among PCV13 serotypes, 19F, 6B and 23F were the top three serotypes while 11A, 15B/C and 8 were the top three among non-PCV13 serotypes in the collection. Two lineages (GPSC6 and GPSC47) expressed both PCV13 and non-PCV13 serotypes that caused invasive disease, and were penicillin- and multidrug-resistant (MDR), highlighting their potential to adapt and continue to cause infections under vaccine and antibiotic selective pressure. PCV13 serotypes comprised 92 % (11/12) of the CSF isolates from the children aged below 5 years; however, the prevalence of PCV13 serotype isolates dropped to 53 % (31/58) among the nasopharyngeal isolates. Our analysis showed that 59 % (105/179) of the isolates were predicted to be non-susceptible to at least one class of antibiotics and 26 % (46/179) were MDR. Four MDR lineages (GPSC1, GPSC6, GPSC10 and GPSC47) accounted for 65 % (30/46) of the MDR isolates and expressed PCV13 serotypes (93 %, 28/30).
This study provides evidence of high genetic and serotype diversity contributed by a mix of globally spreading and regionally circulating lineages in Russia. The observations suggest that the PCV13 vaccine could be important in reducing both invasive disease and antimicrobial resistance. We also identify potential lineages (GPSC6 and GPSC47) that may evade the vaccine.
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Emerging patterns of fluoroquinolone resistance in Campylobacter jejuni in the UK [1998–2018]
More LessCampylobacter jejuni (C.jejuni) is the most common causative agent of bacterial food poisoning worldwide and is known to be genetically highly diverse. C. jejuni is increasingly resistant to fluoroquinolone antibiotics, but very few studies have investigated variant-specific patterns of resistance across time. Here we use statistical modelling and clustering techniques to investigate patterns of fluoroquinolone resistance amongst 10,359 UK isolates from human disease sampled over 20 years. We observed six distinct patterns of fluoroquinolone sensitivity/resistance in C. jejuni across time, grouping by clonal complex (CC). Some CCs were fully resistant, some shifted from susceptible to resistant following a sigmoidal shape, and some remained susceptible over time. Our findings indicate that the fluoroquinolone resistance patterns of C. jejuni are complicated and cannot be analysed as a single species but divided into variant dynamics so that the factors driving resistance can be thoroughly investigated.
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Exploring the mobilome and resistome of Enterococcus faecium in a One Health context across two continents
Haley Sanderson, Kristen L. Gray, Alexander Manuele, Finlay Maguire, Amjad Khan, Chaoyue Liu, Chandana Navanekere Rudrappa, John H. E. Nash, James Robertson, Kyrylo Bessonov, Martins Oloni, Brian P. Alcock, Amogelang R. Raphenya, Tim A. McAllister, Sharon J. Peacock, Kathy E. Raven, Theodore Gouliouris, Andrew G. McArthur, Fiona S. L. Brinkman, Ryan C. Fink, Rahat Zaheer and Robert G. BeikoEnterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, virulence factors and MGEs were profiled in a set of 1273 E. faecium genomes from two disparate geographic locations (in the UK and Canada) from a range of agricultural, clinical and associated habitats. Neither lineages of E. faecium , type A and B, nor MGEs are constrained by geographic proximity, but our results show evidence of a strong association of many profiled genes and MGEs with habitat. Many features were associated with a group of clinical and municipal wastewater genomes that are likely forming a new human-associated ecotype within type A. The evolutionary dynamics of E. faecium make it a highly versatile emerging pathogen, and its ability to acquire, transmit and lose features presents a high risk for the emergence of new pathogenic variants and novel resistance combinations. This study provides a workflow for MGE-centric surveillance of AMR in Enterococcus that can be adapted to other pathogens.
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IS26-mediated plasmid reshuffling results in convergence of toxin–antitoxin systems but loss of resistance genes in XDR Klebsiella pneumoniae from a chronic infection
Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA, a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB–IncHI1B (harbouring armA and bla NDM-1), an IncFIA (bla CTX-M-15) and a ColE-like (bla OXA-232), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA, resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin–antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15, and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS26-mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.
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- Short Communications
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- Pathogens and Epidemiology
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Phylodynamic analysis of SARS-CoV-2 spread in Rio de Janeiro, Brazil, highlights how metropolitan areas act as dispersal hubs for new variants
Alessandra Pavan Lamarca, Luiz G. P. de Almeida, Ronaldo da Silva Francisco Junior, Liliane Cavalcante, Otávio Brustolini, Alexandra L. Gerber, Ana Paula de C. Guimarães, Thiago Henrique de Oliveira, Érica Ramos dos Santos Nascimento, Cintia Policarpo, Isabelle Vasconcellos de Souza, Erika Martins de Carvalho, Mario Sergio Ribeiro, Silvia Carvalho, Flávio Dias da Silva, Marcio Henrique de Oliveira Garcia, Leandro Magalhães de Souza, Cristiane Gomes Da Silva, Caio Luiz Pereira Ribeiro, Andréa Cony Cavalcanti, Claudia Maria Braga de Mello, Amilcar Tanuri and Ana Tereza R. de VasconcelosDuring the first semester of 2021, all of Brazil has suffered an intense wave of COVID-19 associated with the Gamma variant. In July, the first cases of Delta variant were detected in the state of Rio de Janeiro. In this work, we have employed phylodynamic methods to analyse more than 1 600 genomic sequences of Delta variant collected until September in Rio de Janeiro to reconstruct how this variant has surpassed Gamma and dispersed throughout the state. After the introduction of Delta, it has initially spread mostly in the homonymous city of Rio de Janeiro, the most populous of the state. In a second stage, dispersal occurred to mid- and long-range cities, which acted as new close-range hubs for spread. We observed that the substitution of Gamma by Delta was possibly caused by its higher viral load, a proxy for transmissibility. This variant turnover prompted a new surge in cases, but with lower lethality than was observed during the peak caused by Gamma. We reason that high vaccination rates in the state of Rio de Janeiro were possibly what prevented a higher number of deaths.
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Naturally occurring Neisseria gonorrhoeae can have large deletions in housekeeping gene abcZ, making them untypable with multilocus sequence typing
The abcZ gene is an essential housekeeping gene in all the Neisseria species. It is one of the seven genes used for multilocus sequence typing (MLST) this genus. It encodes the cytosolic component of an ATP-binding cassette (ABC) transporter complex of unknown function. We report here the finding of a strain of Neisseria gonorrhoeae with a 485 base pair deletion in the 5′ region of the abcZ gene that truncates the protein product from 636 amino acids to 89 amino acids. A second open reading frame (ORF), encoding the latter 388 amino acids of the abcZ gene, was predicted downstream. The deletion will affect MLST profiling; interrogation of genomic sequences from PubMLST revealed that this isolate is not an anomaly. Deletions in abcZ were identified in 256 Neisseria genomes, roughly 0.6% of isolates. Furthermore, these deletions could leave the abcZ gene in a pseudogenized state. Our strain, isolated from a patient with symptoms of gonorrheal infection, nevertheless behaved normal in terms of growth and in vitro phenotypic properties.
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- Methods
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- Genomic Methodologies
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dRNASb: a systems biology approach to decipher dynamics of host-pathogen interactions using temporal dual RNA-seq data
Infection triggers a dynamic cascade of reciprocal events between host and pathogen wherein the host activates complex mechanisms to recognise and kill pathogens while the pathogen often adjusts its virulence and fitness to avoid eradication by the host. The interaction between the pathogen and the host results in large-scale changes in gene expression in both organisms. Dual RNA-seq, the simultaneous detection of host and pathogen transcripts, has become a leading approach to unravelling complex molecular interactions between the host and the pathogen and is particularly informative for intracellular organisms. The amount of in vitro and in vivo dual RNA-seq data is rapidly growing, which demands computational pipelines to effectively analyse such data. In particular, holistic, systems-level, and temporal analyses of dual RNA-seq data are essential to enable further insights into the host-pathogen transcriptional dynamics and potential interactions. Here, we developed an integrative network-driven bioinformatics pipeline, dRNASb, a systems biology-based computational pipeline to analyse temporal transcriptional clusters, incorporate molecular interaction networks (e.g. protein-protein interactions), identify topologically and functionally key transcripts in host and pathogen, and associate host and pathogen temporal transcriptome to decipher potential between-species interactions. The pipeline is applicable to various dual RNA-seq data from different species and experimental conditions. As a case study, we applied dRNASb to analyse temporal dual RNA-seq data of Salmonella -infected human cells, which enabled us to uncover genes contributing to the infection process and their potential functions and to identify putative associations between host and pathogen genes during infection. Overall, dRNASb has the potential to identify key genes involved in bacterial growth or host defence mechanisms for future uses as therapeutic targets.
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