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- Karpati, F, et al.
(författare)
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Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedish patients with cystic fibrosis reveal genetic heterogeneity
- 2001
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - 0903-4641 .- 1600-0463. ; 109:5, s. 389-400
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Tidskriftsartikel (refereegranskat)abstract
- To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequencing could be used as rapid methods for epidemiological typing and species identification of clinical Burkholderia isolates from patients with cystic fibrosis (CF), a total of 39 clinical B. cepacia isolates, including 33 isolates from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP-PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considerable similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosomal small subunit RNA gene. The multiple sequence alignment produced 12 different patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at least two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease at follow-up. Both the sequencing and the AP-PCR patterns revealed genetic heterogeneity of isolates between patients. According to the results obtained, AP-PCR could advantageously be used for epidemiological typing of Burkholderia, whereas partial species identification could effectively be obtained by sequencing of the V3 region of the 16S RNA gene.
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- Kolak, M., et al.
(författare)
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Molecular typing of the bacterial flora in sputum of cystic fibrosis patients
- 2003
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Ingår i: International Journal of Medical Microbiology. - : Elsevier BV. - 1438-4221 .- 1618-0607. ; 293:4, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA PyrosequencingTM. DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine.
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- Schmidt, I K, et al.
(författare)
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Long-term manipulation of the microbes and microfauna of two subarctic heaths by addition of fungicide, bactericide, carbon and fertilizer
- 2000
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Ingår i: Soil Biology & Biochemistry. - 0038-0717. ; 32:5, s. 707-720
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Tidskriftsartikel (refereegranskat)abstract
- Nutrient availability is a major constraint to plant production and carbon storage in arctic ecosystems, but there are few studies coupling processes in the decomposer and microbial food web and the implications these processes have on the control of nutrient mineralization. We studied the relationship between microbial biomass and the abundance of microbivore and the role of grazing on nutrient turnover after annual addition of carbon (sucrose), fertilizer (NPK), fungicide (benomyl) and bactericides (streptomycin and penicillin) to two dwarf shrub communities, a low and a high altitude heath. After four years of repeated additions, we measured microbial biomass by fumigation-extraction and phospholipid fatty acid (PLFA) analysis, the fungal to bacterial biomass ratio by PLFA analysis and estimated the numbers of protozoa and nematodes, assigned into feeding groups. The fungal to bacterial ratio of nematode feeding groups was around 0.2, indicating a bacterial-based food web in both communities. The size of the microbial biomass did not change after the additions, except when the amount of available carbon was increased (low altitude heath) or when addition of carbon was combined with fertilizer (high altitude heath). In contrast, fertilizer but not carbon increased the number of microbivores. This suggests that the amount of available carbon and not grazing pressure controls the size of the microbial biomass, Furthermore, it suggests that the food quality, e.g. nutrient content of the micro-organisms, had a larger effect on the microbivore than the size of the microbial biomass. The addition of bactericides and fungicide did not significantly change the fungal to bacterial biomass ratio of the micro-organisms. We could not detect any effects of the bactericides. In contrast, the fungicide strongly decreased nematode density, least in the fungal feeders, probably due to increased abundance of the insensitive Aphelenchoides ssp. (C) 2000 Elsevier Science Ltd. All rights reserved.
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