RESUMO
A novel microarray was constructed with DNA PCR product probes targeting species specific functional genes of nine clinically significant respiratory pathogens, including the Gram-positive organisms (Streptococcus pneumoniae, Streptococcus pyogenes), the Gram-negative organisms (Chlamydia pneumoniae, Coxiella burnetii Haemophilus spp., Legionella pneumophila, Moraxella catarrhalis, and Pseudomonas aeruginosa), as well as the atypical bacterium, Mycoplasma pneumoniae. In a "proof-of-concept" evaluation of the developed microarray, the microarray was compared with real-time PCR from 14 sputum specimens from COPD patients. All of the samples positive for bacterial species in real-time PCR were also positive for the same bacterial species using the microarray. This study shows that a microarray using PCR probes is a potentially useful method to monitor the populations of bacteria in respiratory specimens and can be tailored to specific clinical needs such as respiratory infections of particular patient populations, including patients with cystic fibrosis and bronchiectasis.
Assuntos
Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Positivas/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doença Pulmonar Obstrutiva Crônica/microbiologia , Infecções Respiratórias/diagnóstico , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pyogenes/isolamento & purificação , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/isolamento & purificação , Coxiella burnetii/genética , Coxiella burnetii/isolamento & purificação , DNA Bacteriano/análise , DNA Bacteriano/genética , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/etiologia , Infecções por Bactérias Gram-Positivas/etiologia , Haemophilus/genética , Haemophilus/isolamento & purificação , Humanos , Legionella pneumophila/genética , Legionella pneumophila/isolamento & purificação , Moraxella catarrhalis/genética , Moraxella catarrhalis/isolamento & purificação , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/isolamento & purificação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Doença Pulmonar Obstrutiva Crônica/complicações , Infecções Respiratórias/etiologia , Sensibilidade e Especificidade , Especificidade da Espécie , Escarro/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pyogenes/genéticaRESUMO
The strain Burkholderia cepacia G4 aerobically mineralized trichloroethene (TCE) to CO(2) over a time period of approximately 20 h. Three biodegradation experiments were conducted with different bacterial optical densities at 540 nm (OD(540)s) in order to test whether isotope fractionation was consistent. The resulting TCE degradation was 93, 83.8, and 57.2% (i.e., 7.0, 16.2, and 42.8% TCE remaining) at OD(540)s of 2.0, 1.1, and 0.6, respectively. ODs also correlated linearly with zero-order degradation rates (1.99, 1.11, and 0.64 micromol h(-1)). While initial nonequilibrium mass losses of TCE produced only minor carbon isotope shifts (expressed in per mille delta(13)C(VPDB)), they were 57.2, 39.6, and 17.0 per thousand between the initial and final TCE levels for the three experiments, in decreasing order of their OD(540)s. Despite these strong isotope shifts, we found a largely uniform isotope fractionation. The latter is expressed with a Rayleigh enrichment factor, epsilon, and was -18.2 when all experiments were grouped to a common point of 42.8% TCE remaining. Although, decreases of epsilon to -20.7 were observed near complete degradation, our enrichment factors were significantly more negative than those reported for anaerobic dehalogenation of TCE. This indicates typical isotope fractionation for specific enzymatic mechanisms that can help to differentiate between degradation pathways.
