Stereoselective reductase-catalysed deoxygenation of sulfoxides in aerobic and anaerobic bacteria

Direct and indirect evidence, Of unexpected stereoselective reductase-catalysed deoxygenations of sulfoxides, was found. The deoxygenations proceeded simultaneously, with the expected dioxygenase-catalysed asymmetric sulfoxidation of sulfides, during some biotransformations with the aerobic bacterium Pseudomonas putida UV4. Stereoselective reductase-catalysed asymmetric deoxygenation of racemic alkylaryl, dialkyl and phenolic sulfoxides was observed, without evidence of the reverse sulfoxidation reaction, using anaerobic bacterial strains. A purified dimethyl sulfoxide reductase, obtained from the intact cells of the anaerobic bacterium Citrobacter braakii DMSO 11, yielded, from the corresponding racemates, enantiopure alkylaryl sulfoxide and thiosulfinate samples.

Antimicrobial activity of fosfomycin and tobramycin in combination against cystic fibrosis pathogens under aerobic and anaerobic conditions

There is a need for new antibiotics or combination of antibiotics that possess activity against increasingly resistant cystic fibrosis (CF) respiratory pathogens such as Pseudomonas aeruginosa and MRSA.

Resistance Development of Cystic Fibrosis Respiratory Pathogens When Exposed to Fosfomycin and Tobramycin Alone and in Combination under Aerobic and Anaerobic Conditions

Although antibiotics from different classes are frequently prescribed in combination to prevent the development of resistance amongst Cystic Fibrosis (CF) respiratory pathogens, there is a lack of data as to the efficacy of this approach. We have previously shown that a 4:1 (w/w) combination of fosfomycin and tobramycin (F:T) has excellent activity against CF pathogens with increased activity under physiologically relevant anaerobic conditions. Therefore, the aim of this study was to determine whether F:T could delay or prevent the onset of resistance compared to either fosfomycin or tobramycin alone under aerobic and anaerobic conditions. The frequency of spontaneous mutants arising following exposure to fosfomycin, tobramycin and F:T was determined for clinical Pseudomonas aeruginosa and MRSA isolates under aerobic and anaerobic conditions. The effect of sub-inhibitory concentrations of fosfomycin, tobramycin and F:T on the induction of resistance was also investigated, with the stability of resistance and fitness cost associated with resistance assessed if it developed. P. aeruginosa and MRSA isolates had a lower frequency of spontaneous mutants to F:T compared to fosfomycin and tobramycin under both aerobic and anaerobic conditions. There was a maximum two-fold increase in F:T MICs when P. aeruginosa and MRSA isolates were passaged in sub-inhibitory F:T for 12 days. In contrast, sequential resistance to fosfomycin and tobramycin developed quickly (n = 3 days for both) after passage in sub-inhibitory concentrations. Once developed, both fosfomycin and tobramycin resistance was stable and not associated with a biological fitness cost to either P. aeruginosa or MRSA isolates. The results of this study suggest that F:T may prevent the development of resistance compared to fosfomycin or tobramycin alone under aerobic and physiologically relevant anaerobic conditions. F:T may be a potential treatment option in CF patients chronically colonised by MRSA and/or P. aeruginosa.

Reduced Bacterial Colony Count of Anaerobic Bacteria Is Associated with a Worsening in Lung Clearance Index and Inflammation in Cystic Fibrosis

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

Use of culture and molecular analysis to determine the effect of antibiotic treatment on microbial community diversity and abundance during exacerbation in patients with cystic fibrosis

Background: Anaerobic bacteria are increasingly regarded as important in cystic fibrosis (CF) pulmonary infection. The aim of this study was to determine the effect of antibiotic treatment on aerobic and anaerobic microbial community diversity and abundance during exacerbations in patients with CF.

Methods: Sputum was collected at the start and completion of antibiotic treatment of exacerbations and when clinically stable. Bacteria were quantified and identified following culture, and community composition was also examined using culture-independent methods.

Results: Pseudomonas aeruginosa or Burkholderia cepacia complex were detected by culture in 24/26 samples at the start of treatment, 22/26 samples at completion of treatment and 11/13 stable samples. Anaerobic bacteria were detected in all start of treatment and stable samples and in 23/26 completion of treatment samples. Molecular analysis showed greater bacterial diversity within sputum samples than was detected by culture; there was reasonably good agreement between the methods for the presence or absence of aerobic bacteria such as P aeruginosa (kappa=0.74) and B cepacia complex (kappa=0.92), but agreement was poorer for anaerobes. Both methods showed that the composition of the bacterial community varied between patients but remained relatively stable in most individuals despite treatment. Bacterial abundance decreased transiently following treatment, with this effect more evident for aerobes (median decrease in total viable count 2.3 x 10(7) cfu/g, p=0.005) than for anaerobes (median decrease in total viable count 3 x 10(6) cfu/g, p=0.046).

Conclusion: Antibiotic treatment targeted against aerobes had a minimal effect on abundance of anaerobes and community composition, with both culture and molecular detection methods required for comprehensive characterisation of the microbial community in the CF lung. Further studies are required to determine the clinical significance of and optimal treatment for these newly identified bacteria.

The effect of slurry storage and anaerobic-digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17-degrees-C than at 4-degrees-C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T90 values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2-5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T90 value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.

Lung Microbiota and Bacterial Abundance in Patients with Bronchiectasis when Clinically Stable and during Exacerbation

RATIONALE: Characterization of bacterial populations in infectious respiratory diseases will provide improved understanding of the relationship between the lung microbiota, disease pathogenesis and treatment outcomes.

OBJECTIVES: To comprehensively define lung microbiota composition during stable disease and exacerbation in bronchiectasis patients.

METHODS: Sputum was collected from patients when clinically stable and before and after completion of antibiotic treatment of exacerbations. Bacterial abundance and community composition were analyzed using anaerobic culture and 16S rDNA pyrosequencing.

MEASUREMENTS AND MAIN RESULTS: In clinically stable patients, aerobic and anaerobic bacteria were detected in 40/40 (100%) and 33/40 (83%) sputum samples, respectively. The dominant organisms cultured were P. aeruginosa (n=10 patients), H. influenzae (n=12), Prevotella (n=18) and Veillonella (n=13). Pyrosequencing generated over 150,000 sequences, representing 113 distinct microbial taxa; the majority of observed community richness resulted from taxa present in low abundance with similar patterns of phyla distribution in clinically stable patients and patients at the onset of exacerbation. Following treatment of exacerbation, there was no change in total (p=0.925), aerobic (p=0.917) or anaerobic (p=0.683) load and only a limited shift in community composition. Agreement for detection of bacteria by culture and pyrosequencing was good for aerobic bacteria such as P. aeruginosa (kappa=0.84) but poorer for other genera including anaerobes. Lack of agreement was largely due to bacteria been detected by pyrosequencing but not by culture.

CONCLUSIONS: A complex microbiota is present in the lungs of bronchiectasis patients which remains stable through treatment of exacerbations suggesting that changes in microbiota composition do not account for exacerbations.

CULTURE AND CULTURE INDEPENDANT IDENTIFICATION OF BACTERIAL COMMUNITIES IN THE CYSTIC FIBROSIS RESPIRATORY TRACT

Introduction and Aims: The identification of complex chronic polymicrobial infections, such as those observed in the cystic fibrosis (CF) airways, are often a diagnostic challenge. Few studies have compared culture-dependent methods with molecular identification making it hard to describe bacterial communities in a comprehensive manner. The aim of the study is to compare four different methods with respect to their similarities and differences in detection of bacteria. Methods: We compared41 sputum samples fromroutine clinical-culture, extended-culture (aerobic and anaerobic), and molecular identification such as Roche 454-FLX Titanium and T-RFLP to assess concurrence between methodologies in detecting bacteria. The agreement between methodologies in detecting either absence or presence of bacterial taxa was assessed by Kappa (κ) statistics. Results: The majority of bacterial taxa identified by culture were also identified with molecular analysis. In total 2, 60, 25, and 179 different bacterial taxa were identified with clinical-culture, extended-culture, T-RFLP and 454-FLX respectively. Clinical-culture, extended-culture and T-RFLP were poor predictors of species richness when compared to 454-FLX (p < 0.0001). Agreement between methods for detecting Pseudomonas sp. and Burkholderia sp. was good with κ ≥ 0.7 [p < 0.0001] and κ ≥ 0.9 [p < 0.0001] respectively. Detection of anaerobic bacteria, such as Prevotella sp. and Veillonella sp., was moderate between extended-culture and 454-FLX with κ = 0.461 [p < 0.0001] and κ = 0.311 [p = 0.032] respectively, and good between T-RFLP and 454-FLX with κ = 0.577 [p < 0.0001] and κ = 0.808 [p < 0.0001] respectively. Agreement between methods for other main bacterial taxa, such as Staphylcoccus sp. and Streptococcus sp., was poor with only a moderate agreement for detection of Streptococcus sp. observed between T-RFLP and 454-FLX (κ = 0.221 [p = 0.024]). Conclusions: This study demonstrates the increased sensitivity culture-independent microbial identification such as the 454-FLX have over clinical-culture, extended-culture and T-RFLP methodologies. The extended-culture detected majority of the most prevalent bacterial taxa associated with chronic colonisation of the CF airways which were also detected by culture-independent methodologies. However, agreement between methods in detecting number of potentially relevant bacteria is largely lacking.

Effect of polyethylenimine, a cell permeabilizer, on the photosensitized destruction of algae by methylene blue and nuclear fast red

The present study reports the effect a cell permeabilizer, polyethylenimine (PEI) has on the photodynamic effect of methylene blue (MB) and nuclear fast red (NFR) in the presence of hydrogen peroxide (H₂O₂). The photosensitized destruction of the algae Chlorella vulgaris under irradiation with visible light is examined. The photodynamic effect was investigated under aerobic and anaerobic conditions. The presence of a permeabilizer during the photosensitized destruction of C. vulgaris does not enhance the activity of the MB, MB/H₂O₂ system or the NFR, NFR/H₂O ₂ system under aerobic conditions. However under anaerobic conditions we have determined that when a cell permeabilizer was added to the MB/H ₂O₂ system, the photosensitized destruction of C. vulgaris proceeded via a combination of Type I and Type II mechanisms. The presence of PEI enforces MB/H₂O₂ to be active toward the destruction of C. vulgaris whether oxygen is present or absent. Under aerobic and anaerobic conditions the activity of NFR was suppressed in the presence of PEI as a result of electrostatic interactions between the photosensitizer and the cell permeabilizer. The decrease in fluorescence recorded is indicative of destruction of the chlorophyll a pigment. 

Photo-dynamic biocidal action of methylene blue and hydrogen peroxide on the cyanobacterium Synechococcus leopoliensis under visible light irradiation

Biofilm growth on stone surfaces is a significant contributing factor to stone biodeterioration. Current market based biocides are hazardous to the environment and to public health. We have investigated the photo-dynamic effect of methylene blue (MB) in the presence of hydrogen peroxide (H₂O₂) on the destruction of the cyanobacterium Synechococcus leopoliensis (S. leopoliensis) under irradiation with visible light. Data presented in this paper illustrate that illumination of S. leopoliensis in the presence of a photosensitiser (MB) and H₂O₂ results in the decomposition of both the cyanobacterium and the photosensitiser. The presence of MB and H₂O₂ affects the viability of the photosensitiser and the cyanobacterium with the fluorescence of both decreasing by 80% over the irradiation time investigated. The photo-dynamic effect was observed under aerobic and anaerobic conditions indicating that oxygen was not necessary for the process. This novel combination could be effective for the remediation of biofilm colonised stone surfaces

WS02.7 Initial and chronic MRSA infection in cystic fibrosis

Objectives Chronic MRSA infection, which affects approximately 26% of CF patients in the USA, is associated with declining lung function and poor outcomes (Dasenbrook, 2010). Anaerobic niches have been described within the CF lung, potentially influencing the virulence of MRSA. This study aims to compare initial and chronic CF MRSA isolates, following aerobic and anaerobic culture. Methods Isolates, obtained from CF sputum at first isolation [“early” (n = 10)] or up to 5 years later, during chronic infection [“late” (n = 15)] were cultured in aerobic and anaerobic conditions. Differences in virulence were compared using the Galleria mellonella infection model. Biofilm formation of each isolate was assessed following staining with crystal violet. Production of Δ-haemolysin (Δ-hly), a surrogate marker for expression of the virulence regulator agr, was determined by haemolysis assay. Results MRSA grown in anaerobic conditions had significantly increased virulence in the G. mellonella model (p = 0.007), increased biofilm formation (p = 0.006) and increased Δ-hly production (p<0.0001). No significant difference between Δ-hly production or biofilm formation were observed between early and late isolates; however late isolates were found to be more virulent in the G. mellonella model (p = 0.0002). Conclusion These results suggest that an anaerobic environment, as found in the CF lung, may increase virulence of MRSA and aid in the establishment of chronic infection. Further clinical studies are required to determine how these phenotypic changes are associated with transition to chronic infection and patient outcome.

Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers

RATIONALE: The role bacteria play in the progression of COPD has increasingly been highlighted in recent years. However, the microbial community complexity in the lower airways of patients with COPD is poorly characterised.

OBJECTIVES: To compare the lower airway microbiota in patients with COPD, smokers and non-smokers.

METHODS: Bronchial wash samples from adults with COPD (n=18), smokers with no airways disease (n=8) and healthy individuals (n=11) were analysed by extended-culture and culture-independent Illumina MiSeq sequencing. We determined aerobic and anaerobic microbiota load and evaluated differences in bacteria associated with the three cohorts. Culture-independent analysis was used to determine differences in microbiota between comparison groups including taxonomic richness, diversity, relative abundance, 'core' microbiota and co-occurrence.

MEASUREMENT AND MAIN RESULTS: Extended-culture showed no difference in total load of aerobic and anaerobic bacteria between the three cohorts. Culture-independent analysis revealed that the prevalence of members of Pseudomonas spp. was greater in the lower airways of patients with COPD; however, the majority of the sequence reads for this taxa were attributed to three patients. Furthermore, members of Bacteroidetes, such as Prevotella spp., were observed to be greater in the 'healthy' comparison groups. Community diversity (α and β) was significantly less in COPD compared with healthy groups. Co-occurrence of bacterial taxa and the observation of a putative 'core' community within the lower airways were also observed.

CONCLUSIONS: Microbial community composition in the lower airways of patients with COPD is significantly different to that found in smokers and non-smokers, indicating that a component of the disease is associated with changes in microbiological status.