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1.
In this study, the epidemiology of Burkholderia cepacia complex (Bcc) recovered from the sputum of 75 patients attending the Genoa Cystic Fibrosis (CF) Center at the Gaslini Children's Hospital (Genoa, Italy) was investigated, and the clinical course of the CF patients infected with the different species and genomovars of Bcc was evaluated. All isolates were analyzed for genomovar status by recA gene polymorphism and subsequently random amplified polymorphic DNA fingerprinting. Burkholderia cenocepacia is the predominant species recovered from the CF patients infected with Bcc at the Genoa CF Center. Of the other eight species comprising the Bcc, only a few isolates belonging to B. cepacia genomovar I, Burkholderia stabilis, and Burkholderia pyrrocinia were found. Of the four recA lineages of B. cenocepacia, most patients were infected by epidemic strains belonging to lineages IIIA and IIID, whereas only a few patients harbored IIIB strains. Patient-to-patient spread of Bcc among CF patients was mostly associated with B. cenocepacia, in particular with strains belonging to recA lineages IIIA and IIID. The mortality of CF patients infected with Bcc at the Genoa CF Center was significantly higher than mortality among CF patients not infected with Bcc. All of the deaths were associated with the presence of B. cenocepacia, except the case of a patient infected with B. cepacia genomovar I. Within B. cenocepacia, infection with epidemic strains belonging to lineages IIIA and IIID was associated with higher rates of mortality than was infection with lineage IIIB strains. No significant differences in lung function, body weight, and mortality rate were observed between patients infected with epidemic strains belonging to either B. cenocepacia IIIA or B. cenocepacia IIID.  相似文献   

2.
The Burkholderia cepacia complex (Bcc) is a group of significant opportunistic respiratory pathogens which affect people with cystic fibrosis. In this study, we sought to ascertain the epidemiology and geographic species distribution of 116 Bcc isolates collected from people with CF in Australia and New Zealand. We performed a combination of recA-based PCR, amplified rDNA restriction analysis (ARDRA), pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR on each isolate. Each Burkholderia cenocepacia isolate was also screened by PCR for the presence of the B. cepacia epidemic strain marker. One hundred and fourteen isolates were assigned to a species using recA-based PCR and ARDRA. B. cenocepacia, B. multivorans and B. cepacia accounted for 45.7%, 29.3% and 11.2% of the isolates, respectively. Strain analysis of B. cenocepacia revealed that 85.3% of the isolates were unrelated. One related B. cenocepacia strain was identified amongst 15 people. Whilst full details of person-to-person contact was not available, all patients attended CF centres in Queensland (Qld) and New South Wales (NSW). Although person-to-person transmission of B. cenocepacia strains has occurred in Australia, the majority of CF-related Bcc infections in Australia and New Zealand are most likely acquired from the environment.  相似文献   

3.
Bacillus species as a group offer several advantages over other bacteria for protection against root pathogens because of their ability to form endospores, and because of the broad-spectrum activity of their antibiotics. The objectives of this work were to determine the ability of strains of Bacillus to inhibit Fusarium verticillioides growth and fumonisin B(1) accumulation in vitro, and to evaluate the ability of the best bacterium for preventing rhizosphere and endorhizosphere colonization by F. verticillioides. Bacterial populations from the maize rhizoplane were obtained, and the capacity of ten Bacillus strains to inhibit fungal growth and fumonisin B(1) accumulation in vitro was assayed. According to these results, B. subtilis CE1 was selected as the best antagonist for testing maize root colonization of F. verticillioides. Bacillus subtilis CE1 at 10(8) and 10(7) CFU ml(-1) inocula was able to reduce rhizoplane and endorhizosphere colonization of F. verticillioides in greenhouse trials. The strain B. subtilis CE1 could be a potential biological control agent against F. verticillioides at the root level.  相似文献   

4.
Gram-negative bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can infect the lungs of cystic fibrosis (CF) patients and can be transmitted among these patients, causing epidemics in the CF community. Lipopolysaccharide (LPS) is an important virulence factor of many gram-negative bacteria, with the O antigen component of LPS being responsible for serotype specificity. The goal of this work was to develop a genetic method of determining the serotype of Bcc isolates based on the conserved gene wbiI. Homologues of wbiI are found in polysaccharide biosynthesis gene clusters in other bacteria. Primers to a conserved region of the Bcc wbiI gene were able to amplify by PCR a single product in 67 of 80 Bcc isolates tested. Sequencing and restriction enzyme digestion of this wbiI PCR product revealed sufficient DNA polymorphisms to distinguish and group various isolates. In five of nine instances, Bcc isolates of a single serotype had a single wbiI restriction fragment length polymorphism (RFLP) pattern, while isolates of the other four serotypes could have multiple wbiI RFLP types. Species determination of the Bcc isolates revealed no obvious correlation between wbiI RFLP type and species. There was also no apparent correlation between wbiI RFLP type and the ability of a single Bcc isolate to infect an individual with CF. However three of five Bcc outbreaks involved isolates with the same wbiI RFLP type, indicating that wbiI RFLP typing may be a useful tool to help track Bcc outbreaks.  相似文献   

5.
Knowledge about the virulence mechanisms of species from the Burkholderia cepacia complex (BCC) is still limited. The genomovar heterogeneity and production of different virulence factors are likely to contribute to the variation in the clinical outcome observed in BCC-infected cystic fibrosis (CF) patients. Therefore, in this study we investigated the genetic polimorphism, the presence of genetic makers associated with virulence and transmissibility in BCC, and the profile of exoenzyme production of 59 BCC isolates obtained from 59 CF patients attending the reference CF centre in Rio de Janeiro, Brazil. The DNA sequence analyses of the recA gene allowed us to identify 40 of these 59 BCC species as being B. cenocepacia, 9 as B. vietnamiensis, 6 as B. multivorans and 4 as B. ambifaria. The assessment of the bacterial genetic polymorphism by PFGE revealed that B. cenocepacia and the B. multivorans isolates belonged to four and two different PFGE profiles with prevalence of two clones, A and B, respectively. All B. vietnamiensis and B. ambifaria belonged to only one PFGE profile (J and E, respectively). None of the isolates exhibited the genetic markers cblA and BCESM, assessed by polymerase chain reaction. In contrast, the profile of enzymatic activity, assessed by phenotypic methods, differed among the BCC species: protease activity was detected only in B. cenocepacia and B. ambifaria isolates, whereas only B. vietnamiensis isolates produced hemolysin. Although the phospholipase C activity was similar among the different species, the level of lipase activity produced by B. multivorans was higher than in the other species. We speculate that the differential characteristics of exoenzyme production may account for the differences in the pathogenic potentials of each BCC species.  相似文献   

6.
In order to investigate the mechanisms by which Burkholderia cepacia complex (Bcc) strains cross the epithelial barrier of the lung and cause septicaemia in a subgroup of Cystic Fibrosis (CF) patients, the invasiveness of four Bcc species have been examined in three lung epithelial cells: A549, 16HBE14o- and Calu-3. The latter two cell lines form polarised monolayers when grown on filters. Invasion of both cell lines by B. multivorans strains was reduced when the cells were grown as tight monolayers compared unpolarised cells, suggesting basolateral receptors are required for the process. In contrast, four B. cenocepacia strains showed comparable invasion of both cell lines irrespective of culture model. All four species of Bcc reduced the TER of Calu-3 monolayers. However, while B. cepacia, B. multivorans and B. stabilis strains readily translocated across the epithelial monolayer, B. cenocepacia translocation was slower. Both B. multivorans and B. cenocepacia altered expression of ZO-1 in Calu-3 cells, but not E-cadherin. Overall, the findings that Bcc strains from four species, which differ greatly in their virulence, have the potential to disrupt tight junctions and to translocate across the epithelium, demonstrates this effect is not exclusive to the most virulent species.  相似文献   

7.
Differences in infection kinetics and host response between Burkholderia multivorans and Burkholderia cenocepacia were demonstrated in a pulmonary infection model in BALB/c mice. B. multivorans persisted in the lung, while B. cenocepacia was cleared. Indirect immunofluorescence and electron microscopy of B. multivorans-infected lungs localized bacteria to macrophages. Clearance of B. cenocepacia was associated with greater interleukin-1beta and neutrophil responses than the responses induced by B. multivorans.  相似文献   

8.
The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans –the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections.  相似文献   

9.
To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.  相似文献   

10.
The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.  相似文献   

11.
Five acid-tolerant thiosulfate-metabolizing bacteria were isolated from acid mine drainage samples from Garubathan, India. 16S rRNA gene analysis revealed that the strains were affiliated with the genus Burkholderia of the class of Betaproteobacteria. Comparative 16S rRNA gene sequence analyses indicated that the strains designated as GAH1 and GAH2 produced a separate phylogenetic branch having Burkholderia pyrrocinia ATCC 51958T (96-98%) as the closest relative. Strains GAH4 and Burkholderia tropica Ppe8T (93%) branched out separately in the phylogenetic tree. Strain GMX2 was most closely related to Burkholderia cepacia ATCC 25417T (99.6%) and Burkholderia vietnamiensis LMG 10929T (99%). Strain GAH5 was most closely related to B. pyrrocinia ATCC 51958T (98%). Oligotrophy has been demonstrated in all AMD strains of Burkholderia spp. All strains showed chemolithoautotrophic and mixotrophic growth in thiosulfate. Furthermore, cell-free extracts of all test strains possessed thiosulfate and sulfite dehydrogenase activities. Phylogenetic analysis of the soxB gene revealed that GAH4 and GAH2 strains formed a novel cluster, Betaproteobacteria II, having highest similarity with Allochromatium vinosum, a member of Gammaproteobacteria II.  相似文献   

12.
13.
This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71 kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.  相似文献   

14.
Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, phenylpyruvate, and 2-phenylacetamide were usable as sole carbon sources by wild type B. cenocepacia K56-2, but not by a PA catabolism-defective mutant. EMSA analysis showed that the binding of PaaR, the negative regulator protein of B. cenocepacia PA catabolism, to PA regulatory DNA could only be relieved by phenylacetyl-Coenzyme A (PA-CoA), but not by any of the putative phenylalanine degradation intermediates. Taken together, our results show that in B. cenocepacia, phenylalanine is catabolized to PA and induces PA catabolism through PA activation to PA-CoA. Thus, PaaR shares the same inducer with PaaX, the regulator of PA catabolism in Escherichia coli, despite belonging to a different protein family.  相似文献   

15.
We demonstrate that all nine species of the Burkholderia cepacia complex can express the mucoid phenotype. A survey of clinical isolates showed that strains of B. cenocepacia, the most virulent species of the complex, are most frequently nonmucoid. Additionally, isolates from patients with chronic infections can convert from mucoid to nonmucoid.  相似文献   

16.
Burkholderia cepacia complex (Bcc) bacteria have gained notoriety as pathogens in cystic fibrosis (CF) because they are difficult to identify and treat, and also have the ability to spread between CF individuals. Of the 17 formally named species within the complex, Burkholderia multivorans and Burkholderia cenocepacia dominate in CF. Multilocus sequence typing has proven to be a very useful tool for tracing the global epidemiology of Bcc bacteria and has shown that B. cenocepacia strains with high transmissibility, such as the ET-12 strain (ST-28) and the Czech strain (ST-32), have spread epidemically within CF populations in Canada and Europe. The majority of research on the molecular pathogenesis of Bcc bacteria has focused on the B. cenocepacia ET-12 epidemic lineage, with gene mutation, genome sequence analysis and, most recently, global gene expression studies shedding considerable light on the virulence and antimicrobial resistance of this pathogen. These studies demonstrate that the ability of B. cenocepacia to acquire foreign DNA (genomic islands, insertion sequences and other mobile elements), regulate gene expression via quorum sensing, compete for iron during infection, and mediate antimicrobial resistance and inflammation via its membrane and surface polysaccharides are key features that underpin the virulence of different strains. With the wealth of molecular knowledge acquired in the last decade on B. cenocepacia strains, we are now in a much better position to develop strategies for the treatment of pathogenic colonization with Bcc and to answer key questions on pathogenesis concerning, for example, the factors that trigger the rapid clinical decline in CF patients.  相似文献   

17.
Screening is a critical step in the discovery of microbial agents that can exert biological control of Fusarium verticillioides at the root level. The objectives of this research were to determine the utility of a niche overlap index to realise the first screening of maize rhizobacterial isolates during different water activities. Studies were conducted to evaluate various methods for second screening with different modes of action. The antifungal activity of bacterial isolates through antibiosis assay was checked and the influence of different isolates on Fusarium verticilliodes growth and fumonisin B(1) was studied. Eleven competitive rhizobacterial isolates (Arthrobacter globiformis RC1, Azotobacter armeniacus RC2, A. armeniacus RC3, A. globiformis RC4, A. globiformis RC5, A. armeniacus RC6, Pseudomonas solanacearum RC7, Bacillus subtilis RC8, B. subtilis RC9, P. solanacearum RC10, B. subtilis RC11) were selected for the studies which followed. All bacteria were able to utilise the widest range of carbon sources and showed the highest niche overlap indices at the water activities tested. All bacterial antagonists reduced fumonisin B(1) production at all levels tested. Isolates belonging to Pseudomonas and Bacillus genera significantly inhibited fumonisin B(1) production, which ranged between 70 and 100%. Also, A. armeniacus RC2 caused important fumonisin B(1) reduction. The results of the present work suggest that A. armeniacus RC2, A. armeniacus RC3, B. subtilis RC8, B. subtilis RC9, B. subtilis RC11, P. solanacearum RC7, and P. solanacearum RC10 could have practical value in the control of F. verticillioides root colonisation. This paper is part of an on-going study to determine their application at the field level.  相似文献   

18.
The Burkholderia cepacia complex (Bcc) organisms remain significant pathogens in patients with cystic fibrosis (CF). This study was performed to evaluate the prevalence, epidemiological characteristics, and presence of molecular markers associated with virulence and transmissibility of the Bcc strains in the National CF Centre in Belgrade, Serbia. The Bcc isolates collected during the four-year study period (2010–2013) were further examined by 16 s rRNA gene, pulsed-field gel electrophoresis of genomic DNA, multilocus sequence typing analysis, and phylogenetic analysis based on concatenated sequence of seven alleles. Fifty out of 184 patients (27.2 %) were colonized with two Bcc species, B. cenocepacia (n?=?49) and B. stabilis (n?=?1). Thirty-four patients (18.5 %) had chronic colonization. Typing methods revealed a high level of similarity among Bcc isolates, indicating a person-to-person transmission or acquisition from a common source. New sequence types (STs) were identified, and none of the STs with an international distribution were found. One centre-specific ST, B. cenocepacia ST856, was highly dominant and shared by 48/50 (96 %) patients colonized by Bcc. This clone was characterized by PCR positivity for both the B. cepacia epidemic strain marker and cable pilin, and showed close genetic relatedness to the epidemic strain CZ1 (ST32). These results indicate that the impact of Bcc on airway colonization in the Serbian CF population is high and virtually exclusively limited to a single clone of B. cenocepacia. The presence of a highly transmissible clone and probable patient-to-patient spread was observed.  相似文献   

19.
The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.  相似文献   

20.
Bacteria degrading quorum sensing (QS) signals have been proposed as biocontrol agents able to quench QS-dependent expression of virulence symptoms caused by Pectobacterium on potato plants. We report here that gamma-caprolactone (GCL) treatment stimulated growth of the native QS-degrading bacterial community in an industrial plant hydroponic system for culturing Solanum tuberosum. Post-GCL treatment, QS-degrading bacteria were mainly identified as Rhodococcus isolates, while Agrobacterium isolates dominated under similar untreated conditions. Most of the assayed Rhodococcus isolates exhibited efficient biocontrol activity for protecting potato tubers. Analytical chemistry approach revealed the rapid degradation of GCL introduced in the plant cultures.  相似文献   

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