Kenneth D. Bruce

Structure and function of the soil microbial community in microhabitats of a heavy metal polluted soil

Abstract Particle-size fractionation of a heavy metal polluted soil was performed to study the influence of environmental pollution on microbial community structure, microbial biomass, microbial residues and enzyme activities in microhabitats of a Calcaric Phaeocem. In 1987, the soil was experimentally contaminated with four heavy metal loads: (1) uncontaminated controls; (2) light (300 ppm Zn, 100 ppm Cu, 50 ppm Ni, 50 ppm V and 3 ppm Cd); (3) medium; and (4) heavy pollution (two- and threefold the light load, respectively). After 10 years of exposure, the highest concentrations of microbial ninhydrin-reactive nitrogen were found in the clay (2–0.1 μm) and silt fractions (63–2 μm), and the lowest were found in the coarse sand fraction (2,000–250 μm). The phospholipid fatty acid analyses (PLFA) and denaturing gradient gel electrophoresis (DGGE) separation of 16S rRNA gene fragments revealed that the microbial biomass within the clay fraction was predominantly due to soil bacteria. In contrast, a high percentage of fungal-derived PLFA 18 : 2ω6 was found in the coarse sand fraction. Bacterial residues such as muramic acid accumulated in the finer fractions in relation to fungal residues. The fractions also differed with respect to substrate utilization: Urease was located mainly in the <2 μm fraction, alkaline phosphatase and arylsulfatase in the 2–63 μm fraction, and xylanase activity was equally distributed in all fractions. Heavy metal pollution significantly decreased the concentration of ninhydrin-reactive nitrogen of soil microorganisms in the silt and clay fraction and thus in the bulk soil. Soil enzyme activity was reduced significantly in all fractions subjected to heavy metal pollution in the order arylsulfatase >phosphatase >urease >xylanase. Heavy metal pollution did not markedly change the similarity pattern of the DGGE profiles and amino sugar concentrations. Therefore, microbial biomass and enzyme activities seem to be more sensitive than 16S rRNA gene fragments and microbial amino-sugar-N to heavy metal treatment.

Characterization of Bacterial Community Diversity in Cystic Fibrosis Lung Infections by Use of 16S Ribosomal DNA Terminal Restriction Fragment Length Polymorphism Profiling

ABSTRACT Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients. Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients. Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth. The species richness and evenness were determined from the T-RF length and volume for the 71 profiles. Species richness was on average 13.3 ± 7.9 per sample and 13.4 ± 6.7 per patient. On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively. The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively. On average, the remaining T-RF bands represented 10.2% of the total profile volume. The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples. However, 18 other T-RF band lengths were dominant in at least one sample. In conclusion, this reveals the enormous complexity of bacteria within the CF lung. Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling

ABSTRACT The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.

Partitioning core and satellite taxa from within cystic fibrosis lung bacterial communities

Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections that lead to death in the majority of cases. The need to maintain lung function in these patients means that characterising these infections is vital. Increasingly, culture-independent analyses are expanding the number of bacterial species associated with CF respiratory samples; however, the potential significance of these species is not known. Here, we applied ecological statistical tools to such culture-independent data, in a novel manner, to partition taxa within the metacommunity into core and satellite species. Sputa and clinical data were obtained from 14 clinically stable adult CF patients. Fourteen rRNA gene libraries were constructed with 35 genera and 82 taxa, identified in 2139 bacterial clones. Shannon–Wiener and taxa-richness analyses confirmed no undersampling of bacterial diversity. By decomposing the distribution using the ratio of variance to the mean taxon abundance, we partitioned objectively the species abundance distribution into core and satellite species. The satellite group comprised 67 bacterial taxa from 33 genera and the core group, 15 taxa from 7 genera (including Pseudomonas (1 taxon), Streptococcus (2), Neisseria (2), Catonella (1), Porphyromonas (1), Prevotella (5) and Veillonella (3)], the last four being anaerobes). The core group was dominated by Pseudomonas aeruginosa. Other recognised CF pathogens were rare. Mantel and partial Mantel tests assessed which clinical factors influenced the composition observed. CF transmembrane conductance regulator genotype and antibiotic treatment correlated with all core taxa. Lung function correlated with richness. The clinical significance of these core and satellite species findings in the CF lung is discussed.

Use of 16S rRNA Gene Profiling by Terminal Restriction Fragment Length Polymorphism Analysis To Compare Bacterial Communities in Sputum and Mouthwash Samples from Patients with Cystic Fibrosis

ABSTRACT The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found.

Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience

Background Culture-independent analysis of the respiratory secretions of people with cystic fibrosis (CF) has identified many bacterial species not previously detected using culture in this context. However, little is known about their clinical significance or persistence in CF airways. Methods The authors characterised the viable bacterial communities in the sputum collected from 14 patients at monthly intervals over 1 year using a molecular community profiling technique—terminal restriction fragment length polymorphism. Clinical characteristics were also collected, including lung function and medications. Ecological community measures were determined for each sample. Microbial community change over time within subjects was defined using ecological analytical tools, and these measures were compared between subjects and to clinical features. Results Bacterial communities were stable within subjects over time but varied between subjects, despite similarities in clinical course. Antibiotic therapy temporarily perturbed these communities which generally returned to pretreatment configurations within 1 month. Species usually considered CF pathogens and those not previously regarded as such exhibited similar patterns of persistence. Less diverse sputum bacterial communities were correlated to lung disease severity and relative abundance of Pseudomonas aeruginosa. Conclusion Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.

Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition

Rationale Despite the potentially important roles for infection in adult non-cystic fibrosis (CF) bronchiectasis disease progression, the bacterial species present in the lower airways of these patients is poorly characterised. Objectives To provide a comprehensive cross-sectional analysis of bacterial content of lower airway samples from patients with non-CF bronchiectasis using culture-independent microbiology. Methods Paired induced sputum and bronchoalveolar lavage samples, obtained from 41 adult patients with non-CF bronchiectasis, were analysed by 16S ribosomal RNA gene pyrosequencing. Assessment of species distribution and dispersal allowed ‘core’ and ‘satellite’ bacterial populations to be defined for this patient group. Microbiota characteristics correlated with clinical markers of disease. Measurement and main results 140 bacterial species were identified, including those associated with respiratory tract infections and opportunistic infections more generally. A group of core species, consisting of species detected frequently and in high abundance, was defined. Core species included those currently associated with infection in bronchiectasis, such as Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae, and many species that would be unlikely to be reported through standard diagnostic surveillance. These included members of the genera Veillonella, Prevotella and Neisseria. The comparative contribution of core and satellite groups suggested a low level of random species acquisition. Bacterial diversity was significantly positively correlated with forced expiratory volume in 1 s (FEV1) and bacterial community composition similarity correlated significantly with FEV1, neutrophil count and Leicester cough score. Conclusions Characteristics of the lower airways microbiota of adult patients with non-CF bronchiectasis correlate significantly with clinical markers of disease severity.

A novel microbiota stratification system predicts future exacerbations in bronchiectasis.

RATIONALE Although airway microbiota composition correlates with clinical measures in non-cystic fibrosis bronchiectasis, these data are unlikely to provide useful prognostic information at the individual patient level. A system enabling microbiota data to be applied clinically would represent a substantial translational advance. OBJECTIVES This study aims to determine whether stratification of patients according to the predominant microbiota taxon can provide improved clinical insight compared with standard diagnostics. METHODS The presence of bacterial respiratory pathogens was assessed in induced sputum from 107 adult patients by culture, quantitative PCR, and, in 96 samples, by ribosomal gene pyrosequencing. Prospective analysis was performed on samples from 42 of these patients. Microbiological data were correlated with concurrent clinical measures and subsequent outcomes. MEASUREMENTS AND MAIN RESULTS Microbiota analysis defined three groups: Pseudomonas aeruginosa dominated (n = 26), Haemophilus influenzae dominated (n = 34), and other taxa dominated (n = 36). Patients with P. aeruginosa- and H. influenzae-dominated communities had significantly worse lung function, higher serum levels of C-reactive protein (CRP), and higher sputum levels of IL-8 and IL-1β. Predominance of P. aeruginosa, followed by Veillonella species, was the best predictor of future exacerbation frequency, with H. influenzae-dominated communities having significantly fewer episodes. Detection of P. aeruginosa was associated with poor lung function and exacerbation frequency, irrespective of analytical strategy. Quantitative PCR revealed significant correlations between H. influenzae levels and sputum IL-8, IL-1β, and serum CRP. Genus richness was negatively correlated with 24-hour sputum weight, age, serum CRP, sputum IL-1β, and IL-8. CONCLUSIONS Stratification of patients with non-cystic fibrosis bronchiectasis on the basis of predominant bacterial taxa is more clinically informative than either conventional culture or quantitative PCR-based analysis. Further investigation is now required to assess the mechanistic basis of these associations.

Potentially pathogenic airway bacteria and neutrophilic inflammation in treatment resistant severe asthma.

Background Molecular microbiological analysis of airway samples in asthma has demonstrated an altered microbiome in comparison to healthy controls. Such changes may have relevance to treatment-resistant severe asthma, particularly those with neutrophilic airway inflammation, as bacteria might be anticipated to activate the innate immune response, a process that is poorly steroid responsive. An understanding of the relationship between airway bacterial presence and dominance in severe asthma may help direct alternative treatment approaches. Objective We aimed to use a culture independent analysis strategy to describe the presence, dominance and abundance of bacterial taxa in induced sputum from treatment resistant severe asthmatics and correlate findings with clinical characteristics and airway inflammatory markers. Methods Induced sputum was obtained from 28 stable treatment-resistant severe asthmatics. The samples were divided for supernatant IL-8 measurement, cytospin preparation for differential cell count and Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling for bacterial community analysis. Results In 17/28 patients, the dominant species within the airway bacterial community was Moraxella catarrhalis or a member of the Haemophilus or Streptococcus genera. Colonisation with these species was associated with longer asthma disease duration (mean (SD) 31.8 years (16.7) vs 15.6 years (8.0), p = 0.008), worse post-bronchodilator percent predicted FEV1 (68.0% (24.0) vs 85.5% (19.7), p = 0.025) and higher sputum neutrophil differential cell counts (median (IQR) 80% (67–83) vs 43% (29–67), p = 0.001). Total abundance of these organisms significantly and positively correlated with sputum IL-8 concentration and neutrophil count. Conclusions Airway colonisation with potentially pathogenic micro-organisms in asthma is associated with more severe airways obstruction and neutrophilic airway inflammation. This altered colonisation may have a role in the development of an asthma phenotype that responds less well to current asthma therapies.

Staphylococcus aureus small-colony variants are independently associated with worse lung disease in children with cystic fibrosis

BACKGROUND Cystic fibrosis (CF) lung disease is associated with diverse bacteria chronically infecting the airways. Slow-growing, antibiotic-resistant mutants of Staphylococcus aureus known as small-colony variants (SCVs) have been isolated from respiratory secretions from European adults and children with CF lung disease using specific but infrequently used culture techniques. Staphylococcus aureus SCVs can be selected either by exposure to specific antibiotics or by growth with another CF pathogen, Pseudomonas aeruginosa. We sought to determine the prevalence, clinical significance, and likely mechanisms of selection of S. aureus SCVs among a US cohort of children with CF. METHODS We performed a 2-year study of 100 children with CF using culture techniques sensitive for S. aureus SCVs, and evaluated associations with clinical characteristics using multivariable regression models. RESULTS Staphylococcus aureus SCV infection was detected among 24% of participants and was significantly associated with a greater drop in lung function during the study (P = .007, adjusted for age and lung function at enrollment). This association persisted after adjusting for infection with other known CF pathogens, including P. aeruginosa and methicillin-resistant S. aureus. Evidence indicated that S. aureus SCVs were likely selected in vivo by treatment with the antibiotic trimethoprim-sulfamethoxazole and possibly by coinfection with P. aeruginosa. CONCLUSIONS Infection with SCV S. aureus was independently associated with worse CF respiratory outcomes in this pediatric cohort. As many clinical microbiology laboratories do not specifically detect S. aureus SCVs, validation and extension of these findings would require widespread changes in the usual laboratory and clinical approaches to these bacteria.