RESUMEN
Clostridioides difficile is an emerging pathogen of One Health significance. Its highly variable genome contains mobile genetic elements (MGEs) such as transposons and prophages that influence its biology. Systematic deletion of each genetic element is required to determine their precise role in C. difficile biology and contribution to the wider mobilome. Here, Tn5397 (21 kb) and Ï027 (56 kb) were deleted from C. difficile 630 and R20291, respectively, using allele replacement facilitated by CRISPR-Cas9. The 630 Tn5397 deletant transferred PaLoc at the same frequency (1 × 10-7) as 630 harboring Tn5397, indicating that Tn5397 alone did not mediate conjugative transfer of PaLoc. The R20291 Ï027 deletant was sensitive to Ï027 infection, and contained two unexpected features, a 2.7 kb remnant of the mutagenesis plasmid, and a putative catalase gene adjacent to the deleted prophage was also deleted. Growth kinetics of R20291 Ï027 deletant was similar to wild type (WT) in rich medium but marginally reduced compared with WT in minimal medium. This work indicates the commonly used pMTL8000 plasmid series works well for CRISPR-Cas9-mediated gene deletion, resulting in the largest deleted locus (56.8 kb) described in C. difficile. Removal of MGEs was achieved by targeting conjugative/integrative regions to promote excision and permanent loss. The deletants created will be useful strains for investigating Tn5397 or Ï027 prophage contribution to host virulence, fitness, and physiology, and a platform for other mutagenesis studies aimed at functional gene analysis without native transposon or phage interference in C. difficile 630 and R20291.
RESUMEN
The diversity of microbial insertion sequences, crucial mobile genetic elements in generating diversity in microbial genomes, needs to be better represented in current microbial databases. Identification of these sequences in microbiome communities presents some significant problems that have led to their underrepresentation. Here, we present a bioinformatics pipeline called Palidis that recognizes insertion sequences in metagenomic sequence data rapidly by identifying inverted terminal repeat regions from mixed microbial community genomes. Applying Palidis to 264 human metagenomes identifies 879 unique insertion sequences, with 519 being novel and not previously characterized. Querying this catalogue against a large database of isolate genomes reveals evidence of horizontal gene transfer events across bacterial classes. We will continue to apply this tool more widely, building the Insertion Sequence Catalogue, a valuable resource for researchers wishing to query their microbial genomes for insertion sequences.
Asunto(s)
Bacterias , Elementos Transponibles de ADN , Humanos , Bacterias/genética , Biología Computacional , Genoma Microbiano , MetagenómicaRESUMEN
The objective of this review was to assess the prevalence of antibiotic resistance genes in the oral cavity and identify mobile genetic elements (MGEs) important in disseminating them. Additionally, to assess if age, geographic location, oral site, bacterial strains and oral disease influence the prevalence of these genes. Three electronic databases (Medline, Embase and the Cochrane Library) were used to search the literature. Journals and the grey literature were also hand searched. English language studies from January 2000 to November 2020 were selected. Primary screening was performed on the titles and abstracts of 1509 articles generated. One hundred and forty-seven full texts were obtained to conduct the second screening with strict inclusion and exclusion criteria. Forty-four final articles agreed with the inclusion criteria. Half of the studies were classed as low quality. tet(M) was the most prevalent gene overall and the conjugative transposon Tn916 the most common MGE associated with antibiotic resistance genes in the oral cavity. In babies delivered vaginally, tet(M) was more prevalent, whilst tet(Q) was more prevalent in those delivered by C-section. Generally, countries with higher consumption of antibiotics had higher numbers of antibiotic resistance genes. Agricultural as well as medical use of antibiotics in a country should always be considered. Between healthy, periodontitis and peri-implantitis subjects, there was no difference in the prevalence of tet(M); however, erm(B), tet(M) and tet(O) were higher in carious active children than the non-carious group. Subjects with poor oral hygiene have more pathogenic bacteria that carry resistance genes compared to those with good oral hygiene. Enterococcus faecalis isolates demonstrated significant tetracycline resistance (tet(M) up to 60% prevalence in samples) and erythromycin resistance (erm(B) up to 61.9% prevalence in samples), periodontal pathogens showed significant beta-lactam resistance with blaZ and cfxA present in up to 90%-97% of samples and the normal oral flora had a high level of erythromycin resistance with mef(A/E) present in 65% of Streptococcus salivarius isolates. The most common resistance gene was tet(M) in root canals, cfxA in subgingival plaque, erm(B) in supragingival plaque and tet(W) in 100% of whole saliva samples. The review highlights that although many studies in this area have been performed, 50% were classed as low quality. We advise the following recommendations to allow firm conclusions to be drawn from future work: the use of large sample sizes, investigate a broad range of antibiotic resistance genes, improved methodologies and reporting to improve the quality of genetic testing in microbiology and randomisation of subject selection.
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Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Bacterias/genética , Niño , Farmacorresistencia Bacteriana/genética , Eritromicina , Humanos , Secuencias Repetitivas Esparcidas/genética , Pruebas de Sensibilidad Microbiana , Boca , PrevalenciaRESUMEN
There has been an explosion of metagenomic data representing human, animal, and environmental microbiomes. This provides an unprecedented opportunity for comparative and longitudinal studies of many functional aspects of the microbiome that go beyond taxonomic classification, such as profiling genetic determinants of antimicrobial resistance, interactions with the host, potentially clinically relevant functions, and the role of mobile genetic elements (MGEs). One of the most important but least studied of these aspects are the MGEs, collectively referred to as the 'mobilome'. Here we elaborate on the benefits and limitations of using different metagenomic protocols, discuss the relative merits of various sequencing technologies, and highlight relevant bioinformatics tools and pipelines to predict the presence of MGEs and their microbial hosts.
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Bacterias/genética , Microbiota , Animales , Bacterias/clasificación , Bacterias/aislamiento & purificación , Humanos , Secuencias Repetitivas Esparcidas , Metagenoma , MetagenómicaRESUMEN
Antibiotic resistance poses an increasing threat to global health. To tackle this problem, the identification of principal reservoirs of antibiotic resistance genes (ARGs) plus an understanding of drivers for their evolutionary selection are important. During a PCR-based screen of ARGs associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a D-alanine-D-alanine ligase (ddl6 and ddl7) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to D-cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6. A SNP was found to be responsible for this difference in resistance phenotype between the two ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and D-cycloserine binding pockets of Ddl.
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Cicloserina/farmacología , Farmacorresistencia Bacteriana/genética , Integrones/genética , Péptido Sintasas/genética , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Treponema denticola/efectos de los fármacosRESUMEN
The global threat of antimicrobial resistance has driven the use of high-throughput sequencing techniques to monitor the profile of resistance genes, known as the resistome, in microbial populations. The human oral cavity contains a poorly explored reservoir of these genes. Here we analyse and compare the resistome profiles of 788 oral cavities worldwide with paired stool metagenomes. We find country and body site-specific differences in the prevalence of antimicrobial resistance genes, classes and mechanisms in oral and stool samples. Within individuals, the highest abundances of antimicrobial resistance genes are found in the oral cavity, but the oral cavity contains a lower diversity of resistance genes compared to the gut. Additionally, co-occurrence analysis shows contrasting ARG-species associations between saliva and stool samples. Maintenance and persistence of antimicrobial resistance is likely to vary across different body sites. Thus, we highlight the importance of characterising the resistome across body sites to uncover the antimicrobial resistance potential in the human body.
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Bacterias/genética , Farmacorresistencia Bacteriana , Intestinos/microbiología , Boca/microbiología , Antibacterianos/farmacología , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodiversidad , Heces/microbiología , Microbioma Gastrointestinal , Humanos , Metagenoma , FilogeniaRESUMEN
The major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island. These strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile, and loss of the phage protection protein CwpV. A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn1-3) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome. Novel cargo regions of 023_CTnT include genes encoding a sortase, putative sortase substrates, lantibiotic ABC transporters and a putative siderophore biosynthetic cluster. We demonstrate the excision of 023_CTnT and sub-elements 023_CTn2 and 023_CTn3 from the genome of RT023 reference strain CD305 and the transfer of 023_CTn3 to a non-toxigenic C. difficile strain, which may have implications for the use of non-toxigenic C. difficile strains as live attenuated vaccines. Finally, we show that the genes within the island are expressed in a regulated manner in C. difficile RT023 strains conferring a distinct "niche adaptation".
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Membrana Celular/metabolismo , Clostridioides difficile/genética , Elementos Transponibles de ADN/genética , Secuencia de Aminoácidos , Anaerobiosis , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Pared Celular/metabolismo , Genes Bacterianos , Islas Genómicas , Humanos , Microbiota/genética , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismoRESUMEN
Integrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation. In this study, the promoter activity of ORF-less GCs, previously recovered from the oral metagenome, was verified by cloning them upstream of a gusA reporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD. This is the first demonstration and detection of promoter activity of ORF-less GCs from Treponema bacteria and the development of an agar plate-based detection system will enable similar studies in other environments.
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Genes , Metagenoma , Boca/microbiología , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas , Pruebas de Enzimas , Glucuronidasa/metabolismo , Humanos , Integrones/genética , Plásmidos/genéticaRESUMEN
Broad host range conjugative plasmids that replicate in Escherichia coli have been widely used to mobilise smaller replicons, bearing their cognate origin of transfer (oriT) into a variety of organisms that are less tractable genetically, such as Clostridium (Clostridioides) difficile. In this work we demonstrated that the oriT region of pMTL9301 (derived from RK2) is not required for transfer between E. coli and C. difficile strains 630Δerm and CD37 and that this oriT-independent transfer is abolished in the presence of DNase when CD37 is the recipient. Transfer to the 630Δerm strain is DNase resistant even without an obvious oriT, when E. coli CA434 is used as a donor and is sensitive to DNase when E. coli HB101 is the donor.
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Clostridioides difficile/genética , Conjugación Genética , Desoxirribonucleasas/metabolismo , Escherichia coli/genética , Plásmidos/genética , ADN/metabolismo , Elementos Transponibles de ADN , Transferencia de Gen HorizontalRESUMEN
Here, we report the draft genome sequence of Eggerthia catenaformis MAR1 isolated during a screen for d-cycloserine-resistant bacteria from the saliva of healthy humans. Analysis of the genome reveals that the strain has the potential to be a human pathogen and carries genes related to virulence and antibiotic resistance.
RESUMEN
The genomes of all sequenced Clostridium difficile isolates contain multiple mobile genetic elements. The chromosomally located pathogenicity locus (PaLoc), encoding the cytotoxins TcdA and TcdB, was previously hypothesized to be a mobile genetic element; however, mobility was not demonstrated. Here we describe the methods used to facilitate and detect the transfer of the PaLoc from a toxigenic strain into non-toxigenic strains of C. difficile. Although the precise mechanism of transfer has not yet been elucidated, a number of controls are described which indicate transfer occurs via a cell-to-cell-mediated conjugation-like transfer mechanism. Importantly, transfer of the PaLoc was shown to occur on large chromosomal fragments of variable sizes, indicating that homologous recombination is likely to be responsible for the insertion events.
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Bacteriófagos/genética , Cromosomas Bacterianos/química , Clostridioides difficile/genética , Conjugación Genética , Elementos Transponibles de ADN , Transferencia de Gen Horizontal , Islas Genómicas , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Bacteriófagos/efectos de los fármacos , Bacteriófagos/metabolismo , Cromosomas Bacterianos/metabolismo , Cromosomas Bacterianos/virología , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/metabolismo , Clostridioides difficile/virología , Cartilla de ADN/química , Cartilla de ADN/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Recombinación Homóloga , Mitomicina/farmacología , Reacción en Cadena de la Polimerasa , Activación Viral/efectos de los fármacosRESUMEN
A composite transposon is a mobile genetic element consisting of two insertion sequences (ISs) flanking a segment of cargo DNA often containing antibiotic resistance (AR) genes. Composite transposons can move as a discreet unit. There have been recently several reports on a novel mechanism of movement of an IS26-based composite transposon through the formation of a translocatable unit (TU), carrying the internal DNA segment of a composite transposon and one copy of a flanking IS. In this study, we determined the presence of composite transposons and TUs in human oral metagenomic DNA using PCR primers from common IS elements. Analysis of resulting amplicons showed four different IS1216 composite transposons and one IS257 composite transposon in our metagenomic sample. As our PCR strategy would also detect TUs, PCR was carried out to detect circular TUs predicted to originate from these composite transposons. We confirmed the presence of two novel TUs, one containing an experimentally proven antiseptic resistance gene and another containing a putative universal stress response protein (UspA) encoding gene. This is the first report of a PCR strategy to amplify the DNA segment on composite transposons and TUs in metagenomic DNA. This can be used to identify AR genes associated with a variety of mobile genetic elements from metagenomes.
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Elementos Transponibles de ADN , Metagenoma , Boca/microbiología , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Femenino , Voluntarios Sanos , Humanos , Masculino , Metagenómica/métodos , Reacción en Cadena de la Polimerasa/métodos , Saliva/citología , Análisis de Secuencia de ADNRESUMEN
Integrons are genetic elements capable of capturing and expressing open reading frames (ORFs) embedded within gene cassettes. They are involved in the dissemination of antibiotic resistance genes (ARGs) in clinically important pathogens. Although the ARGs are common in the oral cavity the association of integrons and antibiotic resistance has not been reported there. In this work, a PCR-based approach was used to investigate the presence of integrons and associated gene cassettes in human oral metagenomic DNA obtained from both the UK and Bangladesh. We identified a diverse array of gene cassettes containing ORFs predicted to confer antimicrobial resistance and other adaptive traits. The predicted proteins include a putative streptogramin A O-acetyltransferase, a bleomycin binding protein, cof-like hydrolase, competence and motility related proteins. This is the first study detecting integron gene cassettes directly from oral metagenomic DNA samples. The predicted proteins are likely to carry out a multitude of functions; however, the function of the majority is yet unknown.
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Integrones/genética , Metagenoma/genética , Microbiota/genética , Saliva/microbiología , Adulto , Anciano , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Bangladesh , ADN Bacteriano/química , ADN Bacteriano/genética , Farmacorresistencia Microbiana/efectos de los fármacos , Farmacorresistencia Microbiana/genética , Femenino , Genes Bacterianos/genética , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Reino Unido , Adulto JovenRESUMEN
Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.
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Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Placa Dental/microbiología , Metagenoma , Neisseria/química , Saliva/microbiología , Aldehído Oxidorreductasas/genética , Secuencia de Aminoácidos , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Bacterias/efectos de los fármacos , Proteínas Bacterianas/aislamiento & purificación , Cromosomas Artificiales Bacterianos , Clonación Molecular , Escherichia coli/genética , Escherichia coli K12/efectos de los fármacos , Vectores Genéticos , Biblioteca Genómica , Humanos , Lacticaseibacillus casei/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis , Plásmidos , Pseudomonas aeruginosa/efectos de los fármacos , Análisis de Secuencia de ADN , Staphylococcus aureus/efectos de los fármacos , Staphylococcus epidermidis/efectos de los fármacosRESUMEN
BACKGROUND: Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections. METHODS: PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29. RESULTS: Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT. CONCLUSION: This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.
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Clostridioides difficile/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Biopelículas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Clorofilidas , Clostridioides difficile/efectos de la radiación , Células HT29 , Humanos , Azul de Metileno/farmacología , Pruebas de Sensibilidad Microbiana , Porfirinas/farmacologíaRESUMEN
Approximately 11% the Clostridium difficile genome is made up of mobile genetic elements which have a profound effect on the biology of the organism. This includes transfer of antibiotic resistance and other factors that allow the organism to survive challenging environments, modulation of toxin gene expression, transfer of the toxin genes themselves and the conversion of non-toxigenic strains to toxin producers. Mobile genetic elements have also been adapted by investigators to probe the biology of the organism and the various ways in which these have been used are reviewed.
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Clostridioides difficile/genética , Secuencias Repetitivas Esparcidas , Adaptación Biológica , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Transferencia de Gen Horizontal , Genoma BacterianoRESUMEN
Clostridium difficile infection (CDI) is now recognised as the main cause of healthcare associated diarrhoea. Over the recent years there has been a change in the epidemiology of CDI with certain related strains dominating infection. These strains have been termed hyper-virulent and have successfully spread across the globe. Many C. difficile strains have had their genomes completely sequenced allowing researchers to build up a very detailed picture of the contribution of horizontal gene transfer to the adaptive potential, through the acquisition of mobile DNA, of this organism. Here, we review and discuss the contribution of mobile genetic elements to the biology of this clinically important pathogen.