Efficacy of species-specific recA PCR tests in the identification of Burkholderia cepacia complex environmental isolates

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

Interaction of Environmental B. Cenocepacia Strains with Cystic Fibrosis and Non-Cystic Fibrosis Bronchial Epithelial Cells in Vitro.

Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infections, 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 LMG16656T strain. Exposure of polarized CFBE41o- or 16HBE14o- cells to the environmental strains resulted in a significant reduction in transepithelial resistance (TER), comparable to 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 to 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 to 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 the cells which express functional CFTR

Genetic relationships among Italian and Mexican maize-rhizosphere Burkholderia cepacia complex (BCC) populations belonging to Burkholderia cenocepacia IIIB and BCC6 group

<p>Abstract</p> <p>Background</p> <p>A close association between maize roots and <it>Burkholderia cepacia </it>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 <it>Burkholderia cenocepacia </it>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.</p> <p>Results</p> <p>The 31 <it>B. cenocepacia </it>IIIB and 65 BCC6 isolates gave rise to 29 and 39 different restriction types (RTs), respectively. Two pairs of isolates of <it>B. cenocepacia </it>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 <it>B. cenocepacia </it>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 <it>B. cenocepacia </it>IIIB and BCC6 suggests an epidemic population structure in which occasional clones emerge and spread.</p> <p>Conclusions</p> <p>Taken together our data demonstrate a wide dispersal of certain <it>B. cenocepacia </it>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.</p

Impact of clonally-related Burkholderia contaminans strains in two patients attending an Italian cystic fibrosis centre. A case report

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

Soil bacterial community response to differences in agricultural management along with seasonal changes in a Mediterranean region

Land-use change is considered likely to be one of main drivers of biodiversity changes in grassland ecosystems. To gain insight into the impact of land use on the underlying soil bacterial communities, we aimed at determining the effects of agricultural management, along with seasonal variations, on soil bacterial community in a Mediterranean ecosystem where different land-use and plant cover types led to the creation of a soil and vegetation gradient. A set of soils subjected to different anthropogenic impact in a typical Mediterranean landscape, dominated by Quercus suber L., was examined in spring and autumn: a natural cork-oak forest, a pasture, a managed meadow, and two vineyards (ploughed and grass covered). Land uses affected the chemical and structural composition of the most stabilised fractions of soil organic matter and reduced soil C stocks and labile organic matter at both sampling season. A significant effect of land uses on bacterial community structure as well as an interaction effect between land uses and season was revealed by the EP index. Cluster analysis of culture-dependent DGGE patterns showed a different seasonal distribution of soil bacterial populations with subgroups associated to different land uses, in agreement with culture-independent T-RFLP results. Soils subjected to low human inputs (cork-oak forest and pasture) showed a more stable bacterial community than those with high human input (vineyards and managed meadow). Phylogenetic analysis revealed the predominance of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes phyla with differences in class composition across the site, suggesting that the microbial composition changes in response to land uses. Taken altogether, our data suggest that soil bacterial communities were seasonally distinct and exhibited compositional shifts that tracked with changes in land use and soil management. These findings may contribute to future searches for bacterial bio-indicators of soil health and sustainable productivity. X Maite Sampedro Pellicer, Affiliation: ENEA (Italian National Agency for New Technologies, Energy and Sustainable Economic Development) Casaccia Research Center, Technical Unit for Sustainable Development and Innovation of Agro-Industrial System, Rome, Italy X Maria Cristiana Papaleo, Affiliation: Laboratory of Microbial and Molecular Evolution, Department of Biology, University of Florence, Florence, Italy X Alessio Mengoni, Affiliation: Laboratory of Microbial and Molecular Evolution, Department of Biology, University of Florence, Florence, Italy X Luigi Ledda, Affiliation: Dipartimento di Agraria, University of Sassari, Sassari, Italy X Renato Fani, Affiliation: Laboratory of Microbial and Molecular Evolution, Department of Biology, University of Florence, Florence, Italy X Anna Benedetti, Affiliation: Consiglio per la Ricerca e la Sperimentazione in Agricoltura - Research Centre for the Soil-Plant System, Rome, Italy X Claudia Dalmastr

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

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

Classification of Actinoplanes sp. ATCC 33076, an actinomycete that produces the glycolipodepsipeptide antibiotic ramoplanin, as Actinoplanes ramoplaninifer sp. nov.

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 (98.71\u200a% similarity) and Actinoplanes atraurantiacus Y16T (98.33\u200a%). 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-C17\u200a:\u200a0 and iso-C16\u200a:\u200a0, followed by iso-C15\u200a:\u200a0 and moderate amounts of anteiso-C15\u200a:\u200a0, iso-C17\u200a:\u200a0 and C18\u200a:\u200a1 \u3c99c. The genomic DNA G+C\u2009content is 71.4\u2009mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA\u2013DNA 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)

Classification of Nonomuraea sp. ATCC 39727, an actinomycete that produces the glycopeptide antibiotic A40926, as Nonomuraea gerenzanensis sp. nov

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 71.2 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA\u2013DNA 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)

Metabolic profiling of Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia isolates from maize rhizosphere

Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia are the Burkholderia cepacia complex (Bcc) species most frequently associated with roots of crop plants. To investigate the ecophysiological diversity of these species, metabolic profiling of maize rhizosphere isolates was carried out by means of the Biolog system, using GN2 and SFN2 plates and different parameters related to optical density (OD). The metabolic profiles produced by the SFN2 and GN2 plates were identical, but the SFN2's narrower range of OD values and significantly longer reaction times made these plates less suitable for differentiation of isolates. Principal component analysis of maximum OD (ODM) and maximum substrate oxidation rate (mu(M)) data generated by GN2 plates allowed the selection of a reduced number of carbon sources. Statistical analysis of ODM values highlighted marked differences between the metabolic profiles of B. cenocepacia and B. ambifaria, whereas metabolic profiles of B. pyrrocinia clustered very often with those of B. cenocepacia. Analysis of the pm parameter resulted in a slightly lower differentiation among the three Bcc species and a higher metabolic diversity within the single species, in particular within B. cenocepacia. Finally, B. cenocepacia and B. pyrrocinia showed generally higher oxidation rates than B. ambifaria on those GN2 substrates that commonly occur in maize root exudates

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%