RESUMEN
Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.
Asunto(s)
Genoma Viral , Enfermedades de las Plantas , Fagos Pseudomonas , Pseudomonas syringae , Pseudomonas syringae/virología , Pseudomonas syringae/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/virología , Fagos Pseudomonas/genética , Filogenia , Variación GenéticaRESUMEN
The honey bee Apis mellifera is exposed to a variety of biotic and abiotic stressors, such as the highly prevalent microsporidian parasite Nosema (Vairimorpha) ceranae and neonicotinoid insecticides. Both can affect honey bee physiology and microbial gut communities, eventually reducing its lifespan. They can also have a combined effect on the insect's survival. The use of bacterial probiotics has been proposed to improve honey bee health, but their beneficial effect remains an open question. In the present study, western honey bees were experimentally infected with N. ceranae spores, chronically exposed to the neonicotinoid thiamethoxam, and/or supplied daily with the homofermentative bacterium Pediococcus acidilactici MA18/5M thought to improve the honey bees' tolerance to the parasite. Deep shotgun metagenomic sequencing allowed the response of the gut microbiota to be investigated with a taxonomic resolution at the species level. All treatments induced significant changes in honey bee gut bacterial communities. Nosema ceranae infection increased the abundance of Proteus mirabilis, Frischella perrara, and Gilliamella apicola and reduced the abundance of Bifidobacterium asteroides, Fructobacillus fructosus, and Lactobacillus spp. Supplementation with P. acidilactici overturned some of these alterations, bringing back the abundance of some altered species close to the relative abundance found in the controls. Surprisingly, the exposure to thiamethoxam also restored the relative abundance of some species modulated by N. ceranae. This study shows that stressors and probiotics may have an antagonistic impact on honey bee gut bacterial communities and that P. acidilactici may have a protective effect against the dysbiosis induced by an infection with N. ceranae.
RESUMEN
Clostridioides difficile, a Gram-positive spore-forming anaerobic bacterium, has rapidly emerged as the leading cause of nosocomial diarrhoea in hospitals. The availability of large numbers of genome sequences, mainly due to the use of next-generation sequencing methods, has undoubtedly shown their immense advantages in the determination of C. difficile population structure. The implementation of fine-scale comparative genomic approaches has paved the way for global transmission and recurrence studies, as well as more targeted studies, such as the PaLoc or CRISPR/Cas systems. In this chapter, we provide an overview of recent and significant findings on C. difficile using comparative genomic studies with implications for epidemiology, infection control and understanding of the evolution of C. difficile.
Asunto(s)
Clostridioides difficile , Clostridioides , Clostridioides difficile/genética , Composición de Base , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADN , GenómicaRESUMEN
Research on individual viruses and phages, as well as viral populations (viromes), is greatly expanding. Phages and viromes are increasingly suspected to have numerous impacts on the ecosystem in which they reside by interacting directly or indirectly with the other organisms present in their environment. In particular, phage communities of the gut microbiota have been associated with a wide range of diseases. However, properly investigating intestinal viromes is still very challenging, both experimentally and analytically. This chapter proposes a simple and reproducible protocol to separate and enrich DNA phage particles from fecal samples, to sequence them, and finally obtain a basic but robust bioinformatic characterization and classification of the global bacteriophage community.
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Bacteriófagos , Virus , Bacteriófagos/genética , Ecosistema , ADN/genética , Heces , Virus/genética , Metagenómica/métodosRESUMEN
The composition and function of the intestinal microbiota are major determinants of human health and are strongly influenced by diet, antibiotic treatment, lifestyle and geography. Nevertheless, we currently have only little data on microbiomes of non-westernized communities. We assess the stool microbiota composition in 59 children aged 2-5 years from the Adadle district of Ethiopia, Somali Regional State. Here, milk and starch-rich food are predominant components of the local diet, where the inhabitants live a remote, traditional agropastoral lifestyle. Microbiota composition, function and the resistome were characterized by both 16S rRNA gene amplicon and shotgun metagenomic sequencing and compared to 1471 publicly available datasets from children living in traditional, transitional, and industrial communities with different subsistence strategies. Samples from the Adadle district are low in Bacteroidaceae, and Prevotellaceae, the main bacterial representatives in the feces of children living in industrialized and non-industrialized communities, respectively. In contrast, they had a higher relative abundance in Streptococcaceae, Bifidobacteriaceae and Erysipelatoclostridiaceae. Further, genes involved in degradation pathways of lactose, D-galactose and simple carbohydrates were enriched. Overall, our study revealed a unique composition of the fecal microbiota of these agropastoral children, highlighting the need to further characterize the fecal bacterial composition of human populations living different lifestyles.
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Microbioma Gastrointestinal , Humanos , Niño , Microbioma Gastrointestinal/genética , ARN Ribosómico 16S/genética , Etiopía , Bacterias/genética , Heces/microbiología , Conducta AlimentariaRESUMEN
IMPORTANCE: The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.
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Bacteriófagos , Bacteriófagos/genética , Antígenos O/genética , Evolución Biológica , BacteriasRESUMEN
Clostridioides difficile produces toxins that damage the colonic epithelium, causing colitis. Variation in disease severity is poorly understood and has been attributed to host factors and virulence differences between C. difficile strains. We test 23 epidemic ST1 C. difficile clinical isolates for their virulence in mice. All isolates encode a complete Tcd pathogenicity locus and achieve similar colonization densities. However, disease severity varies from lethal to avirulent infections. Genomic analysis of avirulent isolates reveals a 69-bp deletion in the cdtR gene, which encodes a response regulator for binary toxin expression. Deleting the 69-bp sequence in virulent R20291 strain renders it avirulent in mice with reduced toxin gene transcription. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile isolates without reducing colonization and persistence. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.
Asunto(s)
Clostridioides difficile , Colitis , Animales , Ratones , Virulencia/genética , Clostridioides difficile/genética , Clostridioides/metabolismo , Genómica , Colitis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
BACKGROUND: Bacteria and their viruses, bacteriophages, are the most abundant entities of the gut microbiota, a complex community of microorganisms associated with human health and disease. In this ecosystem, the interactions between these two key components are still largely unknown. In particular, the impact of the gut environment on bacteria and their associated prophages is yet to be deciphered. RESULTS: To gain insight into the activity of lysogenic bacteriophages within the context of their host genomes, we performed proximity ligation-based sequencing (Hi-C) in both in vitro and in vivo conditions on the 12 bacterial strains of the OMM12 synthetic bacterial community stably associated within mice gut (gnotobiotic mouse line OMM12). High-resolution contact maps of the chromosome 3D organization of the bacterial genomes revealed a wide diversity of architectures, differences between environments, and an overall stability over time in the gut of mice. The DNA contacts pointed at 3D signatures of prophages leading to 16 of them being predicted as functional. We also identified circularization signals and observed different 3D patterns between in vitro and in vivo conditions. Concurrent virome analysis showed that 11 of these prophages produced viral particles and that OMM12 mice do not carry other intestinal viruses. CONCLUSIONS: The precise identification by Hi-C of functional and active prophages within bacterial communities will unlock the study of interactions between bacteriophages and bacteria across conditions (healthy vs disease). Video Abstract.
Asunto(s)
Bacteriófagos , Profagos , Ratones , Humanos , Animales , Profagos/genética , Ecosistema , Bacteriófagos/genética , Genómica , Cromosomas , Bacterias/genéticaRESUMEN
Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, buthe lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but available data are still limited. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by ÏCD38-2, ÏCD111, and ÏCD146, which normally infect the parental strain. Complementation with 12 different S-layer cassette types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by ÏCD111 and SLCT-11 enabled infection by ÏCD38-2 and ÏCD146. Of note, the expression of SLCT-1, -6, -8, -9, -10, or -12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain. Also, deletion of the D2 domain within the low-molecular-weight fragment of SlpA was found to abolish infection by ÏCD38-2 and ÏCD146 but not ÏCD111. Altogether, our data suggest that many phages use SlpA as their receptor and, most importantly, that both siphophages and myophages target SlpA despite major differences in their tail structures. Our study therefore represents an important step in understanding the interactions between C. difficile and its phages. IMPORTANCE Phage therapy represents an interesting alternative to treat Clostridioides difficile infections because, contrary to antibiotics, most phages are highly species specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage receptor-binding protein binds to a specific SLCT, the identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.
RESUMEN
Clostridioides difficile (C. difficile) , a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences between C. difficile strains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1) C. difficile clinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88 -/- or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of the cdtR gene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-pair cdtR sequence in the highly virulent ST1 R20291 C. difficile strain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile strain without reducing colonization and persistence in the gut. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.
RESUMEN
Viruses that infect bacteria (phages) are increasingly recognized for their importance in diverse ecosystems but identifying and annotating them in large-scale sequence datasets is still challenging. Although efficient scalable virus identification tools are emerging, defining the exact ends (termini) of phage genomes is still particularly difficult. The proper identification of termini is crucial, as it helps in characterizing the packaging mechanism of bacteriophages and provides information on various aspects of phage biology. Here, we introduce PhageTermVirome (PTV) as a tool for the easy and rapid high-throughput determination of phage termini and packaging mechanisms using modern large-scale metagenomics datasets. We successfully tested the PTV algorithm on a mock virome dataset and then used it on two real virome datasets to achieve the rapid identification of more than 100 phage termini and packaging mechanisms, with just a few hours of computing time. Because PTV allows the identification of free fully formed viral particles (by recognition of termini present only in encapsidated DNA), it can also complement other virus identification softwares to predict the true viral origin of contigs in viral metagenomics datasets. PTV is a novel and unique tool for high-throughput characterization of phage genomes, including phage termini identification and characterization of genome packaging mechanisms. This software should help researchers better visualize, map and study the virosphere. PTV is freely available for downloading and installation at https://gitlab.pasteur.fr/vlegrand/ptv .
Asunto(s)
Bacteriófagos/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Secuencia de Empaquetamiento Viral , Viroma , Algoritmos , Bacteriófagos/fisiología , Biología Computacional/métodos , Bases de Datos Genéticas , Metagenómica/métodos , Programas Informáticos , Flujo de TrabajoRESUMEN
Toxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.
Asunto(s)
Clostridioides difficile/genética , Secuencias Repetitivas Esparcidas , Sistemas Toxina-Antitoxina/genética , Regulación Bacteriana de la Expresión Génica , PlásmidosRESUMEN
The epidemiology of Clostridioides difficile infection (CDI) has drastically changed since the emergence of the epidemic strain BI/NAP1/027, also known as ribotype 027 (R027). However, the relationship between the infecting C. difficile strain and clinical outcomes is still debated. We hypothesized that certain subpopulations of R027 isolates could be associated with unfavorable outcomes. We applied high-resolution multilocus variable-number tandem-repeat analysis (MLVA) to characterize C. difficile R027 isolates collected from confirmed CDI patients recruited across 10 Canadian hospitals from 2005 to 2008. PCR ribotyping was performed first to select R027 isolates that were then analyzed by MLVA (n = 450). Complicated CDI (cCDI) was defined by the occurrence of any of admission to an intensive care unit, colonic perforation, toxic megacolon, colectomy, and if CDI was the cause or contributed to death within 30 days after enrollment. Three major MLVA clusters were identified, MC-1, MC-3, and MC-10. MC-1 and MC-3 were exclusive to Quebec centers, while MC-10 was found only in Ontario. Fewer cases infected with MC-1 developed cCDI (4%) than those infected with MC-3 and MC-10 (15% and 16%, respectively), but a statistically significant difference was not reached. Our data did not identify a clear association between subpopulations of R027 and different clinical outcomes; however, the data confirmed the utility of MLVA's higher discrimination potential to better characterize CDI populations in an epidemiological analysis. For a patient with CDI, the progression toward an unfavorable outcome is a complex process that probably includes several interrelated strain and host characteristics.
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Clostridioides difficile/clasificación , Infecciones por Clostridium/epidemiología , Repeticiones de Minisatélite , Anciano , Anciano de 80 o más Años , Técnicas de Tipificación Bacteriana , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/microbiología , Heces/microbiología , Femenino , Hospitalización/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Tipificación de Secuencias Multilocus , Ontario/epidemiología , Quebec/epidemiología , RibotipificaciónRESUMEN
Clostridium difficile, a gram-positive spore-forming anaerobic bacterium, has rapidly emerged as the leading cause of nosocomial diarrhoea in hospitals. The availability of genome sequences in large numbers, mainly due to the use of next-generation sequencing methods, have undoubtedly shown their immense advantages in the determination of the C. difficile population structure. The implementation of fine-scale comparative genomic approaches have paved the way to global transmission and recurrence studies, but also more targeted studies such as the PaLoc or the CRISPR/Cas systems. In this chapter, we provide an overview of the recent and significant findings on C. difficile using comparative genomics studies with implication for the epidemiology, infection control and understanding of the evolution of C. difficile.
Asunto(s)
Clostridioides difficile/genética , Genómica , Sistemas CRISPR-Cas/genética , Infecciones por Clostridium/epidemiología , Infecciones por Clostridium/genética , Infecciones por Clostridium/microbiología , Interacciones Huésped-Patógeno/genética , Humanos , FilogeniaRESUMEN
Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting Lactococcus lactis, and phage c-st, infecting Clostridium botulinum A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen.
Asunto(s)
Clostridioides difficile/genética , Variación Genética , Genoma Viral/genética , Profagos/genética , Clostridioides difficile/patogenicidad , Clostridioides difficile/virología , ADN Viral , Aptitud Genética , Genoma Bacteriano , Genómica/métodos , Humanos , Tipificación de Secuencias Multilocus , Prevalencia , Análisis de Secuencia de ADN , VirulenciaRESUMEN
The worrying rise of antibiotic resistance in pathogenic bacteria is leading to a renewed interest in bacteriophages as a treatment option. Novel sequencing technologies enable description of an increasing number of phage genomes, a critical piece of information to understand their life cycle, phage-host interactions, and evolution. In this work, we demonstrate how it is possible to recover more information from sequencing data than just the phage genome. We developed a theoretical and statistical framework to determine DNA termini and phage packaging mechanisms using NGS data. Our method relies on the detection of biases in the number of reads, which are observable at natural DNA termini compared with the rest of the phage genome. We implemented our method with the creation of the software PhageTerm and validated it using a set of phages with well-established packaging mechanisms representative of the termini diversity, i.e. 5'cos (Lambda), 3'cos (HK97), pac (P1), headful without a pac site (T4), DTR (T7) and host fragment (Mu). In addition, we determined the termini of nine Clostridium difficile phages and six phages whose sequences were retrieved from the Sequence Read Archive. PhageTerm is freely available (https://sourceforge.net/projects/phageterm), as a Galaxy ToolShed and on a Galaxy-based server (https://galaxy.pasteur.fr).
Asunto(s)
Bacteriófagos/fisiología , Genoma Viral , Ensamble de Virus , Animales , Bacteriófagos/clasificación , Clostridioides difficile/virología , ADN ViralRESUMEN
BACKGROUND: Clostridium difficile infection (CDI) is the most common cause of nosocomial infectious diarrhea and may result in severe complications including death. We conducted a prospective study to identify risk factors for complications of CDI (cCDI). METHODS: Adult inpatients with confirmed CDI in 10 Canadian hospitals were enrolled and followed for 90 days. Potential risk factors were measured within 24 hours of diagnosis. Isolates were typed by polymerase chain reaction ribotyping. cCDI was defined as 1 or more of the following: colonic perforation, toxic megacolon, colectomy, admission to an intensive care unit for cCDI, or if CDI contributed to death within 30 days of enrollment. Risk factors for cCDI were investigated by logistic regression. RESULTS: A total of 1380 patients were enrolled. cCDI was observed in 8% of patients. The ribotype was identified in 922 patients, of whom 52% were infected with R027. Age ≥ 80 years, heart rate >90/minute, respiratory rate >20/minute, white cell count <4 × 10(9)/L or ≥ 20 × 10(9)/L, albumin <25 g/L, blood urea nitrogen >7 mmol/L, and C-reactive protein ≥ 150 mg/L were independently associated with cCDI. A higher frequency of cCDI was observed among R027-infected patients (10.9% vs 7.2%), but the association was not significant in adjusted analysis. CONCLUSIONS: CDI complications were associated with older age, abnormal blood tests, and abnormal vital signs. These factors, which are readily available to clinicians at the time of diagnosis, could be used for outcome prediction and risk stratification to select patients who may need closer monitoring or more aggressive therapy.
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Clostridioides difficile/aislamiento & purificación , Cuidados Críticos , Enterocolitis Seudomembranosa/complicaciones , Enterocolitis Seudomembranosa/mortalidad , Perforación Intestinal/epidemiología , Megacolon Tóxico/epidemiología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Canadá , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Ribotipificación , Medición de Riesgo , Adulto JovenRESUMEN
Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Clostridioides difficile/virología , Myoviridae/aislamiento & purificación , Profagos/aislamiento & purificación , Siphoviridae/aislamiento & purificación , Animales , Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , Clostridioides difficile/aislamiento & purificación , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/veterinaria , Dermatoglifia del ADN , ADN Viral/química , ADN Viral/genética , Perros , Electroforesis en Gel de Campo Pulsado , Caballos , Especificidad del Huésped , Humanos , Peso Molecular , Myoviridae/crecimiento & desarrollo , Myoviridae/fisiología , Myoviridae/ultraestructura , Profagos/crecimiento & desarrollo , Profagos/fisiología , Profagos/ultraestructura , Mapeo Restrictivo , Siphoviridae/crecimiento & desarrollo , Siphoviridae/fisiología , Siphoviridae/ultraestructuraRESUMEN
BACKGROUND: Sporulation of Clostridium difficile during infection and persistence of spores within the gut could partly explain treatment failures and recurrence. However, the influence of antibiotics on sporulation is unclear. The objective of our study was to evaluate the impact of ciprofloxacin, metronidazole, piperacillin/tazobactam, tigecycline, and vancomycin on C. difficile sporulation in vitro. METHODS: The reference strains ATCC 9689, 630, VPI 10463, and seven other clinical isolates of C. difficile were used, including three epidemic NAP1/027 isolates. Minimum inhibitory concentrations (MIC) were determined and sporulation was assessed after growth in the absence or presence of ≤0.5x MIC concentrations of each antibiotic. RESULTS: All strains were sensitive to the antibiotics tested, except ribotype 027 isolates that were resistant to ciprofloxacin (MIC = 128 mg/L). Metronidazole and vancomycin generally did not significantly affect spore production in C. difficile, although vancomycin slightly affected sporulation of a few isolates. Ciprofloxacin inhibited sporulation of ribotype 027 isolates mainly. Interestingly, sub-MIC concentrations of piperacillin/tazobactam reduced spore formation in several isolates. However, the most striking observation was made with tigecycline, with an important reduction of spore formation in most isolates. CONCLUSIONS: The capacity of C. difficile to sporulate can be significantly affected by certain antibiotics. The reduced sporulation observed with tigecycline and piperacillin/tazobactam might explain why these antibiotics are generally associated with lower risk of C. difficile infections. In addition, the inhibition of sporulation might partly explain the apparent efficacy of tigecycline for treatment of patients with recurrent infection.