RESUMEN
The filamentous actinomycete that produces the antibiotic GE23077 was isolated by the Lepetit Research Group from a soil sample collected in Thailand, and it was classified as a member of the genus Actinomadura on the basis of its morphology and cell-wall composition. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain formed a distinct monophyletic line within the genus Actinomadura, and it was most closely related to Actinomadura bangladeshensis DSM 45347T (99.31â% similarity) and Actinomadura mexicana DSM 44485T (98.94â%). The GE23077-producing strain formed an extensively branched, non-fragmented vegetative mycelium; no pseudosporangia were formed and the arthrospores were organized in slightly twisted chains. The cell wall contained meso-2,6-diaminopimelic acid and the diagnostic sugar was madurose. The predominant menaquinone was MK-9(H6), with minor amounts of MK-9(H8) and MK-9(H4). The diagnostic phospholipids were phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were C16â:â0 and tuberculostearic acid (10-methyloctadecanoic acid), followed by minor amounts of C18:1ω9c, C16:1ω7c and 10-methylheptadecanoic acid. The genomic DNA G+C content was 71.77 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, and the low DNA-DNA relatedness between the GE23077-producing strain and closely related type strains clearly demonstrate that it represents a novel species of the genus Actinomadura, for which the name Actinomadura lepetitiana sp. nov. is proposed. The type strain is NRRL B-65521T(=LMG 31258T=DSM 109019T).
Asunto(s)
Actinobacteria/clasificación , Filogenia , Microbiología del Suelo , Actinobacteria/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Tailandia , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
BACKGROUND: Burkholderia contaminans is one of the 20 closely related bacterial of the Burkholderia cepacia complex, a group of bacteria that are ubiquitous in the environment and capable of infecting people with cystic fibrosis (CF). This species is an emerging pathogen and it has been widely isolated from CF patients in Argentina, Spain, Portugal, Australia, Canada, USA with a low prevalence in Ireland, France, Russia, Switzerland, Czech Republic, and Italy. This is the first report of B. contaminans affecting two Italian CF patients attending the same CF Centre. We correlate B. contaminans colonisation with lung function decline and co-infection with other clinically relevant CF pathogens. CASE PRESENTATION: B. contaminans was identified by Multi Locus Sequence Typing in routine sputum analysis of two Caucasian CF women homozygous for Phe508del CFTR mutation. Sequence Type 102 was detected in both strains. It is known that B. contaminans ST102 was isolated both from CF and non-CF patients, with an intercontinental spread across the world. Random Amplified Polymorphic DNA analysis revealed the genetic relatedness between the two strains. We examined their susceptibility to antimicrobial agents, comparing the latter with that recorded for other B. contaminans isolated from different countries. We also described key virulence factors possibly linked with a clinical outcome. Specifically, we attempted to correlate colonization with the incidence of acute exacerbation of symptoms and lung function decline. CONCLUSIONS: This case presentation suggests that acquisition of B. contaminans ST102 is not directly associated with a lung function decline. We retain that the presence of other CF pathogens (i.e. MRSA and Trichosporon) along with B. contaminans ST102 might have contributed to the worsening of clinical conditions in our CF patients. The circumstances leading to the establishment of B. contaminans ST102 infections are still unknown. We highlight the importance to proper detect and typing bacteria implicated in CF infection by using molecular techniques.
Asunto(s)
Infecciones por Burkholderia/complicaciones , Complejo Burkholderia cepacia/aislamiento & purificación , Fibrosis Quística/complicaciones , Adulto , Infecciones por Burkholderia/microbiología , Femenino , Humanos , Italia , Pulmón/diagnóstico por imagen , Pulmón/fisiopatología , Tipificación de Secuencias Multilocus , Esputo/microbiología , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Lettuce is a leafy vegetable that is extensively commercialized as a ready-to-eat product because of its widespread use in human nutrition as salad. It is well known that washing treatments can severely affect the quality and shelf-life of ready-to-eat vegetables. The study presented here evaluated the effect of two washing procedures on fresh-cut lettuce during storage. RESULTS: An omics approach was applied to reveal global changes at molecular level induced by peracetic acid washing in comparison with sodium hypochlorite treatment. Microbiological analyses were also performed to quantify total bacterial abundance and composition. The study revealed wide metabolic alterations induced by the two sanitizers. In particular, transcriptomic and proteomic analyses pointed out a number of transcripts and proteins differentially accumulated in response to peracetic acid washing, mainly occurring on the first day of storage. In parallel, different microbiota composition and significant reduction in total bacterial load following washing were also observed. CONCLUSION: The results provide useful information for the fresh-cut industry to select an appropriate washing procedure preserving fresh-like attributes as much as possible during storage of the end product. Molecular evidence indicated peracetic acid to be a valid alternative to sodium hypochlorite as sanitizer solution. © 2017 Society of Chemical Industry.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Lactuca/metabolismo , Ácido Peracético/farmacología , Hipoclorito de Sodio/farmacología , Electroforesis en Gel Bidimensional/métodos , Lactuca/efectos de los fármacos , Espectrometría de Masas/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Longitud del Fragmento de Restricción , Proteómica/métodos , TranscriptomaRESUMEN
Strain ATCC 33076, which produces the antibiotic ramoplanin, was isolated from a soil sample collected in India, and it was classified as a member of the genus Actinoplanes on the basis of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain forms a distinct clade within the genus Actinoplanes, and it is most closely related to Actinoplanes deccanensis IFO 13994T (98.71â% similarity) and Actinoplanes atraurantiacus Y16T (98.33â%). The strain forms an extensively branched substrate mycelium; the sporangia are formed very scantily and are globose with irregular surface. Spores are oval and motile. The cell wall contains meso-diaminopimelic acid and the diagnostic sugars are xylose and arabinose. The predominant menaquinone is MK-9(H6), with minor amounts of MK-9(H4) and MK-9(H2). Mycolic acids are absent. The diagnostic phospholipids are phosphatidylethanolamine, hydroxyphosphatidylethanolamine and phosphatidylglycerol. The major cellular fatty acids are anteiso-C17â:â0 and iso-C16â:â0, followed by iso-C15â:â0 and moderate amounts of anteiso-C15â:â0, iso-C17â:â0 and C18â:â1ω9c. The genomic DNA G+C content is 71.4 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 33076 and closely related type strains, clearly demonstrated that strain ATCC 33076 represents a novel species of the genus Actinoplanes, for which the name Actinoplanes ramoplaninifer sp. nov. is proposed. The type strain is ATCC 33076T (=DSM 105064T=NRRL B-65484T).
Asunto(s)
Depsipéptidos/biosíntesis , Micromonosporaceae/clasificación , Filogenia , Microbiología del Suelo , Técnicas de Tipificación Bacteriana , Composición de Base , Pared Celular/química , ADN Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Grasos/química , India , Micromonosporaceae/genética , Micromonosporaceae/aislamiento & purificación , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
Rectangular DNA origami functionalized with thiols in each of the four corners immobilizes by self-assembly between lithographically patterned gold nanodots on a silicon oxide surface.
Asunto(s)
ADN/química , Oro/química , Nanopartículas del Metal/química , Conformación de Ácido Nucleico , ADN/ultraestructura , Nanopartículas del Metal/ultraestructura , Microscopía de Fuerza AtómicaRESUMEN
Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173T (98.72 % similarity) and Nonomuraea jabiensis A4036T (98.69 %). The strain forms an extensively branched substrate mycelium and aerial hyphae that form spiral chains of spores with ridged surfaces. The cell wall contains meso-diaminopimelic acid and the whole-cell sugars are glucose, ribose, galactose, mannose and madurose (madurose as the diagnostic sugar). The N-acyl type of muramic acid is acetyl. The predominant menaquinone is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H0). The polar-lipid profile includes diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylmethylethanolamine, phosphatidylinositol and a series of uncharacterized phospholipids, glycolipids and phosphoglycolipids. The major cellular fatty acids are iso-C16 : 0 and 10-methyl C17 : 0. The genomic DNA G+C content is 71.2âmol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA-DNA relatedness between strain ATCC 39727 and closely related type strains, clearly demonstrated that strain ATCC 39727 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea gerenzanensis sp. nov. is proposed. The type strain is ATCC 39727T ( = DSM 100948T).
Asunto(s)
Actinomycetales/clasificación , Filogenia , Microbiología del Suelo , Teicoplanina/análogos & derivados , Actinomycetales/genética , Actinomycetales/aislamiento & purificación , Antibacterianos/biosíntesis , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Grasos/química , Glucolípidos/química , India , Ácidos Murámicos/química , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Teicoplanina/biosíntesis , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infection, so it is important to evaluate the pathogenic potential of environmental B. cenocepacia isolates. In this study, we investigated the interactions of two environmental B. cenocepacia strains (Mex1 and MCII-168) with two bronchial epithelial cell lines, 16HBE14o(-) and CFBE41o(-), which have a non-CF and a CF phenotype, respectively. The environmental strains showed a significantly lower level of invasion into both CF and non-CF cells in comparison with the clinical B. cenocepacia LMG16656(T) strain. Exposure of polarized CFBE41o(-) or 16HBE14o(-) cells to the environmental strains resulted in a significant reduction in transepithelial resistance (TER), comparable with that observed following exposure to the clinical strain. A different mechanism of tight junction disruption in CF versus non-CF epithelia was found. In the 16HBE41o(-) cells, the environmental strains resulted in a drop in TER without any apparent effect on tight junction proteins such as zonula occludens-1 (ZO-1). In contrast, in CF cells, the amount of ZO-1 and its localization were clearly altered by the presence of both the environmental strains, comparable with the effect of LMG16656. This study demonstrates that even if the environmental strains are significantly less invasive than the clinical strain, they have an effect on epithelial integrity comparable with that of the clinical strain. Finally, the tight junction regulatory protein ZO-1 appears to be more susceptible to the presence of environmental strains in CF cells than in cells which express a functional cystic fibrosis transmembrane regulator (CFTR).
Asunto(s)
Infecciones por Burkholderia/patología , Burkholderia cenocepacia/patogenicidad , Fibrosis Quística/microbiología , Células Epiteliales/microbiología , Bronquios/citología , Línea Celular , Humanos , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Rizosfera , Uniones Estrechas/microbiología , Zea mays/microbiología , Proteína de la Zonula Occludens-1RESUMEN
BACKGROUND: A close association between maize roots and Burkholderia cepacia complex (BCC) bacteria has been observed in different locations globally. In this study we investigated by MultiLocus Restriction Typing (MLRT) the genetic diversity and relationships among Burkholderia cenocepacia IIIB and BCC6 populations associated with roots of maize plants cultivated in geographically distant countries (Italy and Mexico), in order to provide new insights into their population structure, evolution and ecology. RESULTS: The 31 B. cenocepacia IIIB and 65 BCC6 isolates gave rise to 29 and 39 different restriction types (RTs), respectively. Two pairs of isolates of B. cenocepacia IIIB and BCC6, recovered from both Italian and Mexican maize rhizospheres, were found to share the same RT. The eBURST (Based Upon Related Sequence Types) analysis of MLRT data grouped all the B. cenocepacia IIIB isolates into four clonal complexes, with the RT-4-complex including the 42% of them, while the majority of the BCC6 isolates (94%) were grouped into the RT-104-complex. These two main clonal complexes included RTs shared by both Italian and Mexican maize rhizospheres and a clear relationship between grouping and maize variety was also found. Grouping established by eBURST correlated well with the assessment using unweighted-pair group method with arithmetic mean (UPGMA). The standardized index of association values obtained in both B. cenocepacia IIIB and BCC6 suggests an epidemic population structure in which occasional clones emerge and spread. CONCLUSIONS: Taken together our data demonstrate a wide dispersal of certain B. cenocepacia IIIB and BCC6 isolates in Mexican and Italian maize rhizospheres. Despite the clear relationship found between the geographic origin of isolates and grouping, identical RTs and closely related isolates were observed in geographically distant regions. Ecological factors and selective pressure may preferably promote some genotypes within each local microbial population, favouring the spread of a single clone above the rest of the recombinant population.
Asunto(s)
Burkholderia cenocepacia/genética , Rizosfera , Microbiología del Suelo , Zea mays/microbiología , Alelos , Burkholderia cenocepacia/aislamiento & purificación , ADN Bacteriano/genética , Variación Genética , Italia , Desequilibrio de Ligamiento , México , Raíces de Plantas/microbiología , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
Given the widespread presence of Burkholderia cenocepacia in the rhizosphere it is important to determine whether rhizosphere strains are pathogenic for cystic fibrosis patients or not. Eighteen B. cenocepacia strains of rhizosphere and clinical origin were typed by multi-locus sequence typing (MLST) analysis and compared for their ability to invade pulmonary epithelial cells and their virulence in a mouse model of airway infection. Although there was great variability, clinical strains were the most invasive in vitro. Almost all the rhizosphere and two clinical strains were defined as non-invasive, six clinical strains as invasive, and two strains of both clinical and environmental origin as indeterminate. Exposure of murine airways to clinical strains caused higher acute mortality than that seen after challenge with rhizosphere strains. Furthermore, both clinical and environmental strains were able to persist in the lungs of infected mice, with no significant differences in bacterial loads and localization 14 days after challenge. DNA dot blot analyses of AHL synthase, porin and amidase genes, which play a role in B. cenocepacia virulence, showed that they were present in B. cenocepacia strains irrespective of their origin. Overall, our results suggest that rhizosphere strains do not differ from clinical strains in some pathogenic traits.
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia/patogenicidad , Fibrosis Quística/complicaciones , Microbiología del Suelo , Animales , Técnicas de Tipificación Bacteriana/métodos , Línea Celular , Recuento de Colonia Microbiana , ADN Bacteriano/química , ADN Bacteriano/genética , Células Epiteliales/microbiología , Genotipo , Humanos , Pulmón/microbiología , Masculino , Ratones , Infecciones del Sistema Respiratorio , Análisis de Secuencia de ADN/métodos , Análisis de Supervivencia , Virulencia , Factores de Virulencia/genéticaRESUMEN
Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.
Asunto(s)
Proteínas Bacterianas/genética , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/genética , Girasa de ADN/genética , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/aislamiento & purificación , Cartilla de ADN , ADN Bacteriano/análisis , ADN Bacteriano/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
We describe here one way to achieve hybrid DNA-inorganic nanostructures on rigid flat insulating substrates. We report methods to prepare rectangular DNA origami and incubate them onto arrays of anchoring gold nanodots either in a static solution or in a microfluidic system. We give details on the design and lithographic methods employed to pattern usable arrays of gold nanoanchors on naturally oxidized silicon wafer chips. Scanning electron and atomic force microscopy methodological details are also given for obtaining the relevant characterizations of the immobilized and ordered DNA origami.
Asunto(s)
ADN/química , Oro/química , Silicio/química , Nanopartículas del Metal/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Nanotecnología/instrumentación , Conformación de Ácido NucleicoRESUMEN
The Burkholderia cepacia complex is a group of nine closely related bacterial species that have useful properties in the natural environment as plant pest antagonists, plant growth promoters and degradative agents of toxic substances. Because these species are human opportunistic pathogens, especially in cystic fibrosis patients, biotechnological applications that involve environmental releases have been severely restricted. Recent progress in understanding the taxonomy, epidemiology and ecology of the B. cepacia complex species has unravelled considerable variability in their pathogenicity and ecological properties, which has set the basis for a reassessment of the risk posed by individual species to human health.
Asunto(s)
Complejo Burkholderia cepacia/metabolismo , Fibrosis Quística/microbiología , Complejo Burkholderia cepacia/genética , Complejo Burkholderia cepacia/patogenicidad , Humanos , Control Biológico de Vectores , FilogeniaRESUMEN
With the growing demand of fresh-cut vegetables, a variety of packaging films are produced specifically to improve safety and quality of the fresh vegetables over the storage period. The aim of our work was to evaluate the influence of different packaging films on the quality of fresh-cut lettuce analyzing changes in bacterial community composition and modifications at the proteome level, by means of culture-dependent/culture-independent methods and differential gel electrophoresis combined with mass spectrometry analysis. Total viable counts indicated the presence of a highly variable and complex microbial flora, around a mean value of 6.26 log10 CFU g(-1). Analysis of terminal-restriction fragment length polymorphism data indicated that bacterial communities changed with packaging films and time, showing differences in community composition and diversity indices between the commercially available package (F) and the new packages (A and C), in the first days after packaging. Also proteomic analysis revealed significant changes, involving proteins related to energy metabolism, photosynthesis, plant defense and oxidative stress processes, between F and A/C packages. In conclusion, microbiological and proteomic analysis have proved to be powerful tools to provide new insights into both the composition of leaf-associated bacterial communities and protein content of fresh-cut lettuce during the shelf-life storage process.
Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Biota , Embalaje de Alimentos , Lactuca/química , Lactuca/microbiología , Proteoma/análisis , Recuento de Colonia Microbiana , Electroforesis , Genómica , Espectrometría de Masas , Técnicas Microbiológicas , Polimorfismo de Longitud del Fragmento de Restricción , ProteómicaRESUMEN
Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.
Asunto(s)
Fibrosis Quística/microbiología , Pulmón/microbiología , Microbiota , Esputo/microbiología , Adolescente , Adulto , Niño , Ecología , Femenino , Humanos , Italia , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Filogenia , Prevotella , Pseudomonas , ARN Ribosómico 16S/genética , Pruebas de Función Respiratoria , Fenómenos Fisiológicos Respiratorios , Análisis de Secuencia de ADN , Staphylococcus , Adulto JovenRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0156807.].
RESUMEN
Burkholderia cepacia complex (Bcc) bacteria are naturally present in the rhizosphere of several crop plants and have been found to antagonize a wide range of important plant pathogens. In this study, we evaluated the effect of the pathogenic fungus Fusarium verticillioides on Bcc populations recovered from the roots of Zea mays plants. Maize plants were cultivated under greenhouse conditions and bacterial colonies were randomly isolated from distinct root portions of Fusarium-treated and control plants. We obtained a total of 120 Bcc isolates which all belonged to the species Burkholderia cenocepacia, a species of the Bcc widely distributed in natural habitats such as the rhizosphere of several crop plants. Results obtained revealed that the presence of the plant pathogen F. verticillioides had an effect at the root colonization level of B. cenocepacia populations, since an increase in indigenous B. cenocepacia bacteria was found in the rhizospheres of maize plants grown in infested soil, compared to the rhizospheres of control plants. The analysis of diversity indices as well as the investigation of genetic polymorphism of B. cenocepacia strains, isolated from Fusarium-treated and control root portions, revealed greater genetic variability in the presence of F. verticillioides, especially in the terminal root system portion. Finally, all B. cenocepacia isolates were also tested for in vitro inhibition of F. verticillioides growth as a functional property. Our results revealed that all B. cenocepacia isolates were able to restrict in vitro fungal growth, suggesting that there was no relationship between genetic polymorphism and biocontrol traits.
Asunto(s)
Antibiosis , Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/crecimiento & desarrollo , Fusarium/crecimiento & desarrollo , Raíces de Plantas/microbiología , Zea mays/microbiología , Complejo Burkholderia cepacia/genética , Recuento de Colonia Microbiana , ADN Bacteriano/análisis , Variación Genética , Haplotipos , Enfermedades de las Plantas/microbiología , Técnica del ADN Polimorfo Amplificado Aleatorio , Suelo/análisis , Microbiología del Suelo , Zea mays/crecimiento & desarrolloRESUMEN
In this study, we evaluated if recA species-specific PCR assays could be successfully applied to identify environmental isolates of the widespread Burkholderia cepacia complex (Bcc) species. A total of 729 Bcc rhizosphere isolates collected in different samplings were assigned to the species B. cepacia genomovar I (61), B. cenocepacia recA lineage IIIB (514), B. ambifaria (124) and B. pyrrocinia (30), by means of recA (RFLP) analysis, and PCR tests were performed to assess sensitivity and specificity of recA species-specific primers pairs. B. cepacia genomovar I specific primers produced the expected amplicon with all isolates of the corresponding species (sensitivity, 100%), and cross-reacted with all B. pyrrocinia isolates. On the contrary, B. cenocepacia IIIB primers did not give the expected amplicon in 164 B. cenocepacia IIIB isolates (sensitivity, 68.1%), and isolates of distinct populations showed different sensitivity. B. ambifaria primers failed to amplify a recA-specific fragment only in a few isolates of this species (sensitivity, 93.5%). The absence of specific amplification in a high number of B. cenocepacia rhizosphere isolates indicates that recA specific PCR assays can lead to an underestimation of environmental microorganisms belonging to this bacterial species.
Asunto(s)
Complejo Burkholderia cepacia/clasificación , Complejo Burkholderia cepacia/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Rec A Recombinasas/genética , Microbiología del Suelo , Zea mays/microbiología , Proteínas Bacterianas/genética , Complejo Burkholderia cepacia/genética , Dermatoglifia del ADN , Raíces de Plantas/microbiología , Polimorfismo de Longitud del Fragmento de Restricción , Sensibilidad y EspecificidadRESUMEN
In this work the analysis of the plasmid presence on soil aerobic cultivable heterotrophic bacterial communities was carried out checking a panel of 1,200 isolates, in order to establish the frequency of plasmid presence as well as the degree of plasmid flow between strains affiliated to the same or different taxon. Bacterial communities were isolated from two different sites of a 13-year experimental field with a clay-silt texture. Plasmid molecules were detected at low frequency (27 isolates, 2%) with a size ranging between 2 Kb and 40 Kb. The RAPD analysis performed on the plasmid-harboring isolates and the phylogenetic analysis of the whole community using the 16S rRNA gene sequences revealed the existence of transfer of the same plasmids between strains belonging to the same species and, in some cases, to different species of the same genus. As it might be expected, even though the viable cells title did not differ significantly between the two samplings, the overall data disclosed an uneven distribution of both species and plasmid-harboring strains.
RESUMEN
Cystic fibrosis (CF) is a genetic disease resulting in chronic polymicrobial infections of the airways and progressive decline in lung function. To gain insight into the underlying causes of severe lung diseases, we aimed at comparing the airway microbiota detected in sputum of CF patients with stable lung function (S) versus those with a substantial decline in lung function (SD). Microbiota composition was investigated by using culture-based and culture-independent methods, and by performing multivariate and statistical analyses. Culture-based methods identified some microbial species associated with a worse lung function, i.e. Pseudomonas aeruginosa, Rothia mucilaginosa, Streptococcus pneumoniae and Candida albicans, but only the presence of S. pneumoniae and R. mucilaginosa was found to be associated with increased severe decline in forced expiratory volume in 1 second (FEV1). Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis revealed a higher bacterial diversity than that detected by culture-based methods. Molecular signatures with a statistically significant odds ratio for SD status were detected, and classified as Pseudomonas, Burkholderia and Shewanella, while for other Terminal Restriction Fragments (T-RFs) no species assignation was achieved. The analysis of T-RFLP data using ecological biodiversity indices showed reduced Evenness in SD patients compared to S ones, suggesting an impaired ecology of the bacterial community in SD patients. Statistically significant differences of the ecological biodiversity indices among the three sub-groups of FEV1 (normal/mild vs moderate vs severe) were also found, suggesting that the patients with moderate lung disease experienced changes in the airway assembly of taxa. Overall, changes in CF airway microbial community associated with a severe lung function decline were detected, allowing us to define some discriminatory species as well as some discriminatory T-RFs that represent good candidates for the development of predictive biomarkers of substantial decline in lung function.
Asunto(s)
Fibrosis Quística/microbiología , Pulmón/microbiología , Adolescente , Adulto , Burkholderia/genética , Burkholderia/aislamiento & purificación , Candida albicans/genética , Candida albicans/aislamiento & purificación , Fibrosis Quística/fisiopatología , Progresión de la Enfermedad , Femenino , Volumen Espiratorio Forzado , Humanos , Pulmón/fisiopatología , Masculino , Microbiota , Tipificación Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Shewanella/genética , Shewanella/aislamiento & purificación , Esputo/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Adulto JovenRESUMEN
Clinical and environmental strains of Burkholderia cenocepacia belonging to the recA lineages IIIA and IIIB were examined for exopolysaccharide (EPS) production. The exopolysaccharides structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy. All the strains produced Cepacian, a highly branched polysaccharide constituted of a heptasaccharide repeating unit, composed of one rhamnose, one glucose, one glucuronic acid, one mannose and three galactose residues. This polymer is the most common exopolysaccharide produced by strains of the Burkholderia cepacia (Bcc) complex. One clinical strain produced also another polysaccharide constituted of three galactose units and one 3-deoxy-D-manno-2-octulosonic acid residues, a polymer that was previously isolated from two strains of B. cepacia genomovar I and B. cenocepacia IIIA.