Bacterial activity in cystic fibrosis lung infections

BACKGROUND: Chronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens. METHODS: Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles. RESULTS: Samples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P ≤ 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients. CONCLUSION: These results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

<p>Abstract</p> <p>Background</p> <p>Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment.</p> <p>Methods</p> <p>Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches.</p> <p>Results</p> <p>A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures.</p> <p>Conclusion</p> <p>Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant.</p

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured

Changes in the chemistry of shallow groundwater related to the 2008 injection of CO2 at the ZERT field site, Bozeman, Montana

Approximately 300 kg/day of food-grade CO2 was injected through a perforated pipe placed horizontally 2–2.3 m deep during July 9–August 7, 2008 at the MSU-ZERT field test to evaluate atmospheric and near-surface monitoring and detection techniques applicable to the subsurface storage and potential leakage of CO2. As part of this multidisciplinary research project, 80 samples of water were collected from 10 shallow monitoring wells (1.5 or 3.0 m deep) installed 1–6 m from the injection pipe, at the southwestern end of the slotted section (zone VI), and from two distant monitoring wells. The samples were collected before, during, and following CO2 injection. The main objective of study was to investigate changes in the concentrations of major, minor, and trace inorganic and organic compounds during and following CO2 injection. The ultimate goals were (1) to better understand the potential of groundwater quality impacts related to CO2 leakage from deep storage operations, (2) to develop geochemical tools that could provide early detection of CO2 intrusion into underground sources of drinking water (USDW), and (3) to test the predictive capabilities of geochemical codes against field data. Field determinations showed rapid and systematic changes in pH (7.0–5.6), alkalinity (400–1,330 mg/l as HCO3), and electrical conductance (600–1,800 μS/cm) following CO2 injection in samples collected from the 1.5 m-deep wells. Laboratory results show major increases in the concentrations of Ca (90–240 mg/l), Mg (25–70 mg/l), Fe (5–1,200 ppb), and Mn (5–1,400 ppb) following CO2 injection. These chemical changes could provide early detection of CO2 leakage into shallow groundwater from deep storage operations. Dissolution of observed carbonate minerals and desorption-ion exchange resulting from lowered pH values following CO2 injection are the likely geochemical processes responsible for the observed increases in the concentrations of solutes; concentrations generally decreased temporarily following four significant precipitation events. The DOC values obtained are 5 ± 2 mg/l, and the variations do not correlate with CO2 injection. CO2 injection, however, is responsible for detection of BTEX (e.g. benzene, 0–0.8 ppb), mobilization of metals, the lowered pH values, and increases in the concentrations of other solutes in groundwater. The trace metal and BTEX concentrations are all significantly below the maximum contaminant levels (MCLs). Sequential leaching of core samples is being carried out to investigate the source of metals and other solutes

Co-parenting Mediates the Influence of Marital Satisfaction on Child Adjustment: The Conditional Indirect Effect by Parental Empathy

This study investigated the mediational role of co-parenting in the association between marital satisfaction and child adjustment, by exploring the conditional indirect effect by parental empathy. Using a sample of 101 Italian father-mother dyads with school-aged children, we administered to parents a series of measures in order to assess marital satisfaction, co-parenting, parents\u2019 empathic skills and children\u2019s adjustment. We computed conditional indirect analyses in order to analyze the mediational role of co-parenting in the associations between marital satisfaction and child adjustment. Consequently, we computed a moderated mediated model in order to explore if mothers\u2019 and fathers\u2019 empathic skills moderated the mediating role of co-parenting. Our findings showed that lower levels of co-parenting mediated the associations between mothers and fathers marital dissatisfaction and children\u2019s behavioral problems. Results also showed that this effect was moderated by parent\u2019s empathic skills, such that mediation is stronger for those with lower empathic competencies (moderated mediation)

Adolescent parents’ values: the role played by retrospective perceptions of the family-of-origin

Family-of-origin is an important system in individual’s lives, and its influence is extensive over the life cycle. The aim of this study was to investigate, in a sample of adolescents’ parents, whether their retrospective perceptions of parenting and relationship qualities within their families-of-origin during adolescence predicted their current collectivist and individualist values. Participants included 110 Portuguese adults with a mean age of 46.86 years old. The results of structural equation modeling supported that retrospective perceptions of parenting and relationship qualities in the family-of-origin predicted the adults’ collectivist values but not their individualist values. This study highlights the importance of retrospective perceptions of supporting and non-rejecting parenting for value acquisition. The results also suggest that values that preserve and protect family connections and collaborations are more likely to be socialized by families. Implications for practice and for the theories on parenting, values, family-of-origin influences and family intergenerational transmission are discussed.This work was supported by the Ph.D. grant of the first author (SFRH/BD/62182/2009), financed by Technology and Science Foundation (FCT)