RESUMO
The term 'shotgun metagenomics' is applied to the direct sequencing of DNA extracted from a sample without culture or target-specific amplification or capture. In diagnostic metagenomics, this approach is applied to clinical samples in the hope of detecting and characterizing pathogens. Here, I provide a conceptual overview, before reviewing several recent promising proof-of-principle applications of metagenomics in virus discovery, analysis of outbreaks and detection of pathogens in contemporary and historical samples. I also evaluate future prospects for diagnostic metagenomics in the light of relentless improvements in sequencing technologies.
Assuntos
Infecções Bacterianas/diagnóstico , Metagenômica , Doenças Parasitárias/diagnóstico , Viroses/diagnóstico , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Parasitos/genética , Parasitos/isolamento & purificação , Análise de Sequência de DNA , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
Although previous bacterial typing methods have been informative about potential relatedness of isolates collected during outbreaks, next-generation sequencing has emerged as a powerful tool to not only look at similarity between isolates, but also put differences into biological context. In this study, we have investigated the whole genome sequence of five Pseudomonas aeruginosa isolates collected during a persistent six-year outbreak at Nottingham University Hospitals National Health Service (NHS) Trust City Campus, United Kingdom. Sequencing, using both Roche 454 and Illumina, reveals that most of these isolates are closely related. Some regions of difference are noted between this cluster of isolates and previously published genome sequences. These include regions containing prophages and prophage remnants such as the serotype-converting bacteriophage D3 and the cytotoxin-converting phage phi CTX. Additionally, single nucleotide polymorphisms (SNPs) between the genomic sequence data reveal key single base differences that have accumulated during the course of this outbreak, giving insight into the evolution of the outbreak strain. Differentiating SNPs were found within a wide variety of genes, including lasR, nrdG, tadZ, and algB. These have been generated at a rate estimated to be one SNP every four to five months. In conclusion, we demonstrate that the single base resolution of whole genome sequencing is a powerful tool in analysis of outbreak isolates that can not only show strain similarity, but also evolution over time and potential adaptation through gene sequence changes.
Assuntos
Surtos de Doenças , Genoma Bacteriano/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Monitoramento Epidemiológico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Hospitais , Humanos , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Polimorfismo de Nucleotídeo Único , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Fatores de Tempo , Reino Unido/epidemiologiaRESUMO
The remarkable success of taxonomic discovery, powered by culturomics, genomics and metagenomics, creates a pressing need for new bacterial names while holding a mirror up to the slow pace of change in bacterial nomenclature. Here, I take a fresh look at bacterial nomenclature, exploring how we might create a system fit for the age of genomics, playing to the strengths of current practice while minimizing difficulties. Adoption of linguistic pragmatism-obeying the rules while treating recommendations as merely optional-will make it easier to create names derived from descriptions, from people or places or even arbitrarily. Simpler protologues and a relaxed approach to recommendations will also remove much of the need for expert linguistic quality control. Automated computer-based approaches will allow names to be created en masse before they are needed while also relieving microbiologists of the need for competence in Latin. The result will be a system that is accessible, inclusive and digital, while also fully capable of naming the unnamed millions of bacteria.
RESUMO
We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Helicobacter/genética , Análise de Sequência de DNA , Animais , Infecções por Helicobacter/microbiologia , Humanos , Dados de Sequência Molecular , Filogenia , Homologia de Sequência , Wolinella/genéticaRESUMO
The development of multidrug-resistantAcinetobacter baumanniiis of serious concern in the hospital setting. Here, we report draft genome sequences of 11A. baumanniiisolates that were isolated from a single patient over a 65-day period, during which time the isolates exhibited increased antimicrobial resistance.
RESUMO
The tricorn protease is an archaeal protease that forms massive proteasome-like capsids with a hollow chamber. beta-Propeller and PDZ domains are thought to play a role in substrate selection. By analysis of predicted proteins from novel bacterial genome sequences, we have identified four new bacterial tricorn-like proteases, complete with similar beta-propeller, PDZ and catalytic domains. We propose various hypotheses as to the function of these domains that can now be tested in the laboratory.
Assuntos
Bactérias/enzimologia , Endopeptidases/genética , Estrutura Terciária de Proteína/genética , Sequência de Aminoácidos , Domínio Catalítico/genética , Endopeptidases/análise , Análise de Sequência de Proteína , Homologia de SequênciaRESUMO
A range of surface proteins is anchored to the cell walls of Gram-positive pathogens such as Staphylococcus aureus by the transpeptidase sortase. Until now, sortase-like proteins and their substrates appeared to be limited mainly to such pathogens. However, by searching for sortase homologues among complete and incomplete genome sequences, we have found them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean. There is usually more than one sortase-like protein encoded in each Gram-positive genome, and the genes encoding the sortase-like proteins are often clustered with genes encoding their likely substrates.
Assuntos
Aminoaciltransferases/metabolismo , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/enzimologia , Sequência de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias , Cisteína Endopeptidases , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
ADP-ribosylation is a post-translational modification that can be seen in many contexts, including as the primary mechanism of action of many important bacterial exotoxins. By data-mining complete and incomplete bacterial genome sequences, we have discovered >20 novel putative ADP-ribosyltransferases, including several new potential toxins.
Assuntos
ADP Ribose Transferases/metabolismo , Bactérias/enzimologia , Toxinas Bacterianas/química , Exotoxinas/química , Sequência de Aminoácidos , Bacillus/química , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/metabolismo , Dados de Sequência Molecular , Mycoplasma pneumoniae/química , Toxina Pertussis , Pseudomonas/química , Salmonella typhi/química , Homologia de Sequência de Aminoácidos , Fatores de Virulência de Bordetella/química , Fatores de Virulência de Bordetella/metabolismoRESUMO
Pandoraea species, in particular Pandoraea apista, are opportunistic, multidrug-resistant pathogens in persons with cystic fibrosis (CF). To aid in understanding the role of P. apista in CF lung disease, we used Illumina MiSeq and nanopore MinION technology to sequence the whole genome of the P. apista LMG 16407(T).
RESUMO
The polymerase chain reaction (PCR) was used to discriminate between toxigenic and non-toxigenic isolates of Corynebacterium diphtheriae. Primers specific to the diphtheria toxin gene were used to amplify a toxin gene fragment from simple boiled-cell preparations. Eight recent clinical isolates and four reference strains were tested. The result of the PCR agreed with the traditional toxigenicity assays (the Elek test and guinea pig inoculation) in all cases. PCR has several advantages over the Elek test: it gives a same-day result, it works on colonies taken from selective media, and it detects the toxin gene in mixed cultures. One potential drawback is that the PCR might give a false positive result with the occasional isolate carrying an inactive toxin gene. The good predictive value of a negative PCR result, however, should make it a valuable screening test.
Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium diphtheriae/classificação , Sequência de Bases , Infecções por Corynebacterium/diagnóstico , Corynebacterium diphtheriae/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
AIMS: To assess the performance of the polymerase chain reaction (PCR) when used to screen rapidly large numbers of corynebacteria for toxin production; and to determine the incidence of false positive PCR results with non-toxigenic Corynebacterium diphtheriae isolates. METHODS: Eighty seven recent British isolates of corynebacteria were assayed by PCR. All isolates were assayed from both blood and tellurite agar within a five day period. Thirty three non-toxigenic isolates of C diphtheriae from six countries were also tested by PCR and by the Elek immunodiffusion assay. RESULTS: There was complete concordance between the results of PCR and traditional methods on the recent British isolates, with one exception: an Elek positive "C ulcerans" isolate, which was PCR positive from tellurite but not from blood agar. One of the thirty three (3%) non-toxigenic isolates of C diphtheriae was PCR positive. CONCLUSIONS: These results suggest that PCR compares favourably with traditional methods for the detection of toxigenic corynebacteria and that it represents a powerful new tool in the diagnosis of an old disease.
Assuntos
Corynebacterium diphtheriae/metabolismo , Toxina Diftérica/biossíntese , Sequência de Bases , Corynebacterium diphtheriae/genética , Difteria/diagnóstico , Reações Falso-Positivas , Humanos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.
Assuntos
Endopeptidases/química , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Dados de Sequência MolecularRESUMO
Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.
Assuntos
DNA Bacteriano/genética , Genes Bacterianos , Reação em Cadeia da Polimerase , Sequência de Aminoácidos , Bactérias/genética , Bactérias/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Clonagem Molecular , Genes Reguladores , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Filogenia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Screening of a Clostridium difficile lambda EMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb HindIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, beta-hydroxybutyryl coenzyme A dehydrogenase and thiolase).
Assuntos
Mapeamento Cromossômico , Clostridioides difficile/genética , Clostridium/genética , Genes Bacterianos/genética , 3-Hidroxiacil-CoA Desidrogenases/genética , Acetil-CoA C-Acetiltransferase/genética , Sequência de Aminoácidos , Sequência de Bases , Butanóis/metabolismo , Butiratos/metabolismo , Ácido Butírico , Cromossomos Bacterianos , Clonagem Molecular , Clostridioides difficile/enzimologia , Clostridium/enzimologia , Clostridium/metabolismo , Enoil-CoA Hidratase/genética , Ácidos Graxos/metabolismo , Biblioteca Genômica , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta/genética , OxirreduçãoRESUMO
With the advent of the polymerase chain reaction (PCR), molecular biology is at last poised to enter the clinical microbiology laboratory. We describe this technique, and review its present and future applications in the diagnosis of infectious disease, with particular emphasis on its potential in diagnostic bacteriology. We discuss the suitability of different sequences as targets for DNA amplification. The disadvantages of PCR as a diagnostic strategy are covered, and current technical problems with the method are surveyed. We briefly mention two alternative strategies--the transcript-based amplification system and replicatable RNA reporter systems based on the Q beta replicase.
Assuntos
Amplificação de Genes , Infecções/microbiologia , Técnicas Microbiológicas , Reação em Cadeia da Polimerase , Humanos , Infecções/epidemiologia , Infecções/genética , Transcrição GênicaRESUMO
Shared care of military and civilian patients has resulted in transmission of multidrug-resistant Acinetobacter baumannii (MDR-Aci) from military casualties to civilians. Current typing technologies have been useful in revealing relationships between isolates of A. baumannii but they are unable to resolve differences between closely related isolates from small-scale outbreaks, where chains of transmission are often unclear. In a recent hospital outbreak in Birmingham, six patients were colonised with MDR-Aci isolates indistinguishable using standard techniques. We used whole-genome sequencing to identify single nucleotide polymorphisms in these isolates, allowing us to discriminate between alternative epidemiological hypotheses in this setting.
Assuntos
Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/classificação , Técnicas de Tipagem Bacteriana , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Genoma Bacteriano , Análise de Sequência de DNA , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Reino Unido/epidemiologiaRESUMO
Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered 'irreducibly complex' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems ('molecular bricolage'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.
Assuntos
Proteínas de Bactérias/metabolismo , Evolução Molecular , Flagelos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Dados de Sequência Molecular , Via Secretória , Homologia de Sequência de AminoácidosRESUMO
The record of guinea-pig inoculations carried out at the London Hospital during a 5-year period was examined. A total of 677 specimens were inoculated into guinea-pigs: 34 were positive on culture and guinea-pig post-mortem and 22 were positive on culture alone. Only five were positive on guinea-pig post-mortem when culture was negative and in only three of these five cases were mycobacteria isolated from the guinea-pig tissues. All three cases had been diagnosed effectively on histological grounds, although in one case the results of sensitivity testing did alter treatment. Despite this marginal benefit it is suggested, on grounds of cost, safety, and animal welfare, that the practice of routine inoculation of suspected tuberculous tissue specimens into guinea-pigs should be discontinued.