Design and manufacture of a lyophilised faecal microbiota capsule formulation to GMP standards.

Faecal microbiota transplant (FMT) is an established and effective treatment for recurrent Clostridioides difficile infection (CDI) and has many other potential clinical applications. However, preparation and quality of FMT is poorly standardised and clinical studies are hampered by a lack of well-defined FMT formulations that meet regulatory standards for medicines. As an alternative to FMT suspensions for administration by nasojejunal tube or colonoscopy, which is invasive and disliked by many patients, this study aimed to develop a well-controlled, standardised method for manufacture of lyophilised FMT capsules and to provide stability data allowing storage for extended time periods. Faecal donations were collected from healthy, pre-screened individuals, homogenised, filtered and centrifuged to remove dietary matter. The suspension was centrifuged to pellet bacteria, which were resuspended with trehalose and lyophilised to produce a powder which was filled into 5 enteric-coated capsules (size 0). Live-dead bacterial cell quantitative PCR assay showed <10 fold viable bacterial load reduction through the manufacturing process. No significant loss of viable bacterial load was observed after storage at -80 °C for 36 weeks (p = 0.24, n = 5). Initial clinical experience demonstrated that the capsules produced clinical cure in patients with CDI with no adverse events reported (n = 7). We provide the first report of a detailed manufacturing protocol and specification for an encapsulated lyophilised formulation of FMT. As clinical trials into intestinal microbiota interventions proceed, it is important to use a well-controlled investigational medicinal product in the studies so that any beneficial results can be replicated in clinical practice.

Deprescribing montelukast in children with asthma: a systematic review.

BACKGROUND: National and international asthma guidelines recommend adjusting asthma treatment based on levels of control, yet no guidance is given regarding the stepping-down of montelukast in children and young people (CYP). OBJECTIVE: To systematically review evidence regarding deprescribing montelukast in CYP with established asthma. DESIGN: Systematic review. DATA SOURCES: Embase, Medline, PubMed and CINAHL were searched up to October 2020. STUDY SELECTION: Eligible studies contained patients aged 0-18 years with a diagnosis of asthma, who had been administering montelukast before it was withdrawn. All reasons for withdrawal were included. RESULTS: The search identified 197 papers. After deduplication, five papers were included (three randomised control studies and two cohort studies). Four studies observed the impact of montelukast withdrawal for 2 weeks, and one study for 8 weeks. The impact of withdrawal was measured in the studies using a combination of lung tests (eg, forced expiratory volume in 1 s (FEV1), fractional exhaled nitric oxide (FeNO)), asthma scoring methods and exercise challenges. Of the 17 domains in the Core Outcome Set for Clinical Trials in Childhood Asthma, eight outcomes were measured in at least one of the five studies, with all five studies measuring the outcome of 'Lung Function'. No significant differences were found between the montelukast and placebo groups following montelukast withdrawal. Significant differences between the comparator points within the test group were found in nine outcomes across four studies; FEV1/forced vital capacity, FEV1, forced expiratory flows (25%-75%), asthma score (study specific), maximum % fall in FEV1 and time to recovery (post exercise) significantly decreased whereas FEV1/bronchodilator response, FeNO and eNO significantly increased. CONCLUSION: Only limited, contradictory and short-term effects of deprescribing montelukast in CYP with established asthma are presented in literature. Definitive studies determining clinical stability, and impact of deprescribing montelukast in CYP are imperative to improve the safety of asthma treatment in CYP. PROSPERO REGISTRATION NUMBER: CRD42020213971.

Porphyromonas pasteri and Prevotella nanceiensis in the sputum microbiota are associated with increased decline in lung function in individuals with cystic fibrosis.

Although anaerobic bacteria exist in abundance in cystic fibrosis (CF) airways, their role in disease progression is poorly understood. We hypothesized that the presence and relative abundance of the most prevalent, live, anaerobic bacteria in sputum of adults with CF were associated with adverse clinical outcomes. This is the first study to prospectively investigate viable anaerobic bacteria present in the sputum microbiota and their relationship with long-term outcomes in adults with CF. We performed 16S rRNA analysis using a viability quantitative PCR technique on sputum samples obtained from a prospective cohort of 70 adults with CF and collected clinical data over an 8 year follow-up period. We examined the associations of the ten most abundant obligate anaerobic bacteria present in the sputum with annual rate of FEV1 change. The presence of Porphyromonas pasteri and Prevotella nanceiensis were associated with a greater annual rate of FEV1 change; -52.3 ml yr-1 (95 % CI-87.7;-16.9), -67.9 ml yr-1 (95 % CI-115.6;-20.1), respectively. Similarly, the relative abundance of these live organisms were associated with a greater annual rate of FEV1 decline of -3.7 ml yr-1 (95 % CI: -6.1 to -1.3, P=0.003) and -5.3 ml yr-1 (95 % CI: -8.7 to -1.9, P=0.002) for each log2 increment of abundance, respectively. The presence and relative abundance of certain anaerobes in the sputum of adults with CF are associated with a greater rate of long-term lung function decline. The pathogenicity of anaerobic bacteria in the CF airways should be confirmed with further longitudinal prospective studies with a larger cohort of participants.

Empowering children and young people who have asthma.

Asthma is the most common chronic condition of childhood. In this review, we discuss an overview of strategies to empower children and young people with asthma. The key aspects of empowerment are to enable shared decision making and self-management, and help children minimise the impact of asthma on their life. The evidence behind these strategies is either sparse or heterogenous, and it is difficult to identify which interventions are most likely to improve clinical outcomes. Wider determinants of health, in high-resource and low-resource settings, can be disempowering for children with asthma. New approaches to technology could help empower young people with asthma and other chronic health conditions.

Selection and assembly of indigenous bacteria and methanogens from spent metalworking fluids and their potential as a starting culture in a fluidized bed reactor.

Waste metalworking fluids (MWFs) are highly biocidal resulting in real difficulties in the, otherwise favoured, bioremediation of these high chemical oxygen deman (COD) wastes anaerobically in bioreactors. We have shown, as a proof of concept, that it is possible to establish an anaerobic starter culture using strains isolated from spent MWFs which are capable of reducing COD or, most significantly, methanogenesis in this biocidal waste stream. Bacterial strains (n = 99) and archaeal methanogens (n = 28) were isolated from spent MWFs. The most common bacterial strains were Clostridium species (n = 45). All methanogens were identified as Methanosarcina mazei. Using a random partitions design (RPD) mesocosm experiment, we found that bacterial diversity and species-species interactions had significant effects on COD reduction but that bacterial composition did not. The RPD study showed similar effects on methanogenesis, except that composition was also significant. We identified bacterial species with positive and negative effects on methane production. A consortium of 16 bacterial species and three methanogens was used to initiate a fluidized bed bioreactor (FBR), in batch mode. COD reduction and methane production were variable, and the reactor was oscillated between continuous and batch feeds. In both microcosm and FBR experiments, periodic inconsistencies in bacterial reduction in fermentative products to formic and acetic acids were identified as a key issue.

Contributions of Composition and Interactions to Bacterial Respiration Are Reliant on the Phylogenetic Similarity of the Measured Community.

Bacterial diversity underpins many ecosystem functions; however, the impact of within-species variation on the relationship between diversity and function remains unclear. Processes involving strain differentiation, such as niche radiation, are often overlooked in studies that focus on phylogenetic variation. This study used bacterial isolates assembled in two comparable microcosm experiments to test how species variation affected ecosystem function. We compared the relationship between diversity and activity (CO2 production) in increasingly diverse multispecies microcosms and with multiple ecotypes of a single species. The bacteria used were isolated from a low-diversity environment and are species of potential clinical significance such as Pseudomonas aeruginosa. All isolates were profiled for single carbon source utilisation. These data showed an increased breadth of resource use in the multiple ecotypes when compared to the mixed-species. The study observed significantly increasing respiration in more complex mixed-species assemblages, which was not observed when ecotypes of a single species were combined. We further demonstrate that the variation observed in the bacterial activity was due to the roles of each of the constituent isolates; between different species, the interactions between the isolates drove the variation in activity, whilst in single species, assemblage variation was due to which isolates were present. We conclude that both between- and within-species variations play different roles in community function, although through different mechanisms, and should be included in models of changing diversity and ecosystem functioning.

Using pharmacokinetic modelling to improve prescribing practices of intravenous aminophylline in childhood asthma exacerbations.

OBJECTIVE: To evaluate physiologically based pharmacokinetic modelling (PBPK) software in paediatric asthma patients using intravenous aminophylline. METHODS: Prospective clinical audit of children receiving iv aminophylline (July 2014 to June 2016), and in-silico modelling using Simcyp software. RESULTS: Thirty-eight admissions (25 children) were included. Children with aminophylline levels ≥10 mg/l had equivalent clinical outcomes compared to those <10 mg/L, and adverse effects occurred in 57%. Therapeutic drug monitoring (TDM) data correlated well with PBPK model. PBPK modelling of a 5 mg/kg iv loading dose (≤18yr) shows a mean Cmax of 8.99 mg/L (5th-95th centiles 5.5-13.7 mg/L), with 70.3% of subjects <10 mg/L, 29.4% achieving 10-20 mg/L, and 0.1% > 20 mg/L. For an aminophylline infusion (0-12 y) of 1.0  mg/kg/h, the mean steady state infusion concentration was 16.4 mg/L, (5th-95th centiles 5.3-32 mg/L), with 26.8% having a serum concentration >20 mg/L. For 12-18yr receiving 0.5  mg/kg/h infusion, the mean steady state infusion concentration was 9.37 mg/L (5th-95th centiles 3.4-18 mg/L), with 59.8% having a serum concentration <10 mg/L. CONCLUSION: PBPK software modelling correlates well with clinical data. Current aminophylline iv loading dosage recommendations achieve levels <10 mg/l in 70% of children. Routine TDM may need altering as low risk of toxicity (>20 mg/l).

ASSESSING THE BENEFITS THAT COMMUNITY PHARMACIES CAN HAVE ON CHILDHOOD ASTHMA OUTCOMES.

INTRODUCTION: The local Clinical Commissioning Group has funded an innovative one-year pilot project to assess the value of providing specialist paediatric pharmacist and physiotherapist support direct to families and health care professionals (GP's, community pharmacists, practice nurses etc.) regarding asthma in the primary care setting. Community pharmacies are the one service that asthmatic children come in contact with in order to pick up their medications it was decided to encourage staff to provide interventions at the point of collection. METHODS: 22 large chain, small chain and independent community pharmacy branches were included in the pilot (out of 152 within CCG area) with a total of 31 pharmacists and 67 assistants trained to provide the service. The plan was to provide 'back to basics' leaflets on collection of prescription to help improve education on the medications being used; provide inhaler technique counselling on the collection of all prescriptions for children; encourage pharmacist's to perform medicines use reviews and the new medicines service in asthmatic children of high school age (for which they could collect the standard NHS fee). In order to assess the benefits of this, the pharmacist or assistant would first perform the standard asthma control test, marked out of 25 with the parent/patient completing an online version one month later to assess any improvement in symptom management. In order to trace the number of MURs, NMS, inhaler counselling sessions and leaflets given out a tally chart was completed each month by the branches involved. RESULTS: Unfortunately of the 22 branches that signed up to the pilot only 15 returned tally charts to the team. Over a six month period 23 MUR's, 3 NMS and 32 inhaler technique sessions were performed with 67 leaflets distributed. Of a possible 55 asthma control tests (MURs and inhaler technique counselling sessions) only 23 patients completed the four week post intervention online form. Of those completed the average asthma control test score increased by 7 points (30% increase). In particular feedback from the pharmacists involved was that the inhaler counselling sessions were of particular benefit to parents/patients.Feedback from the pharmacy teams in general was positive with many stating it was good to be more involved in the care of children's conditions; however many stated in order for the service to roll out to a wider audience the scheme would have to provide a financial incentive for the large chains to take part. CONCLUSIONS: It is clear that interventions performed by the community pharmacy teams can help improve symptom control in asthmatic children. In particular ensuring patients are using their medications correctly appears to be key to symptom control. Encouraging pharmacists to provide child friendly MURs should be investigated further to prove the benefit of this service further. It should be noted that ensuring patients are using their medications correctly is already part of the essential service contract for pharmacies.

ASSESSING THE BENEFITS OF HAVING A SPECIALIST PAEDIATRIC PHARMACIST AND PHYSIOTHERAPIST IN THE COMMUNITY TO IMPROVE CHILDHOOD ASTHMA OUTCOMES.

INTRODUCTION: The local Clinical Commissioning Group has funded an innovative one-year pilot project to assess the value of providing specialist paediatric pharmacist and physiotherapist support direct to families and health care professionals (GP's, community pharmacists, practice nurses etc.) regarding asthma in the primary care setting. Currently no such support is provided within community setting by physiotherapy or pharmacy. METHODS: Joint holistic reviews by the clinical specialist physiotherapist and specialist paediatric pharmacist were performed in the patient's home environment or school. The review involved a thorough respiratory review and in-depth medication optimisation review ensuring patients were on appropriate regimes and using devices appropriately. Specifically, if an inhaler was indicated a device that the patient was comfortable using was chosen. Furthermore, parents, patients, teachers and school support workers were counselled on how to self manage asthma exacerbations. In order to review benefits patients answered the five question asthma control test (score out of 25), a standardised quality of life questionnaire (score out of 92) and hospital admissions were monitored. RESULTS: At the six-month stage of the project a total of 42 patients had been reviewed and followed up by the project. During the review period there was a total of 1 hospital admission and 1 attendance to the accident and emergency, this is in comparison to the 8 hospital admissions and 47 accident and emergency attendances with this group of patients in the same period the previous year. All patients had an improvement in outcome measures. The average improvement in asthma control test after intervention was 7 points (30%) and a 30% increase in QoL score. We found that symptomatic children had poor FEF25-75 values (<80%) indicating poor lower airways function possibly due to poor drug deposition. After interventions these scores returned to normal limits (>80%). Compliance to medications regimes was noted to be improved after optimisation. CONCLUSIONS: It can be clearly seen that joint multidisciplinary reviews by physiotherapy and pharmacy can help improve the outcomes of asthma patients. The joint review of inhaler technique in particular was key. Pharmacy services will tend to concentrate on the use of the device itself whereas physiotherapy monitor the strength and depth of breathes taken. It is well known that good drug deposition is key to the success of inhaled medications. By combining pharmacy and physiotherapy both the use of the device and breathing patterns were optimised both contributing to better drug deposition and improved FEF 25-75% reading. Optimisation of medication was also vital. By ensuring the patient was happy with the device they were using and that it had little negative impact on their daily routine compliance with medications was increased. At the six month stage of the pilot this aspect has proven vital to outcomes in asthmatic patients.

Environmentally co-occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context-dependent fitness effects.

Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co-occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid-borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus-encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co-occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.

Reducing bias in bacterial community analysis of lower respiratory infections.

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing 'core' and 'satellite' taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.

Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation?

BACKGROUND: Cystic Fibrosis (CF) lung disease is characterised by an inexorable decline in lung function, punctuated by periods of symptomatic worsening known as pulmonary exacerbations (referred to here as CFPE). Despite their clinical significance, the cause of CFPE remains undetermined. It has been suggested that an increase in bacterial density may be a trigger, although this has not been shown empirically. METHODS: Here, a previously validated quantitative PCR-based approach was used to assess numbers of Pseudomonas aeruginosa and of total bacteria in respiratory secretions from patients during the period leading up to CFPE. Sputum samples collected from 12 adult CF patients were selected retrospectively to fall approximately 21, 14, 7 and 0 days prior to CFPE diagnosis. In addition, the relationships between clinical parameters (FEV(1), temperature and patient reported outcome measures) and microbiological data were investigated. RESULTS: No significant changes either in total bacterial or P. aeruginosa numbers were identified prior to CFPE. Of all the correlations tested, only temperature showed a significant correlation with total bacterial numbers in the period leading to CFPE. CONCLUSIONS: These findings strongly suggest that CFPE do not generally result from increased bacterial density within the airways. Instead, data presented here are consistent with alternative models of pulmonary exacerbation.

Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers.

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

Determining the effects of a spatially heterogeneous selection pressure on bacterial population structure at the sub-millimetre scale.

A key interest of microbial ecology is to understand the role of environmental heterogeneity in shaping bacterial diversity and fitness. However, quantifying relevant selection pressures and their effects is challenging due to the number of parameters that must be considered and the multiple scales over which they act. In the current study, a model system was employed to investigate the effects of a spatially heterogeneous mercuric ion (Hg(2+)) selection pressure on a population comprising Hg-sensitive and Hg-resistant pseudomonads. The Hg-sensitive bacteria were Pseudomonas fluorescens SBW25::rfp and Hg-resistant bacteria were P. fluorescens SBW25 carrying a gfp-labelled, Hg resistance plasmid. In the absence of Hg, the plasmid confers a considerable fitness cost on the host, with µ(max) for plasmid-carrying cells relative to plasmid-free cells of only 0.66. Two image analysis techniques were developed to investigate the structure that developed in biofilms about foci of Hg (cellulose fibres imbued with HgCl(2)). Both techniques indicated selection for the resistant phenotype occurred only in small areas of approximately 178-353 µm (manually defined contour region analysis) or 275-350 µm (daime analysis) from foci. Hg also elicited toxic effects that reduced the growth of both Hg-sensitive and Hg-resistant bacteria up to 250 µm from foci. Selection for the Hg resistance phenotype was therefore highly localised when Hg was spatially heterogeneous. As such, for this model system, we define here the spatial scale over which selection operates. The ability to quantify changes in the strength of selection for particular phenotypes over sub-millimetre scales is useful for understanding the scale over which environmental variables affect bacterial populations.

Anthropogenic disturbance affects the structure of bacterial communities.

Patterns of taxa abundance distributions are the result of the combined effects of historical and biological processes and as such are central to ecology. It is accepted that a taxa abundance distribution for a given community of animals or plants following a perturbation will typically change in structure from one of high evenness to increasing dominance. Subsequently, such changes in evenness have been used as indicators of biological integrity and environmental assessment. Here, using replicated experimental treehole microcosms perturbed with different concentrations of the pollutant pentachlorophenol, we investigated whether changes in bacterial community structure would reflect the effects of anthropogenic stress in a similar manner to larger organisms. Community structure was visualized using rank-abundance plots fitted with linear regression models. The slopes of the regression models were used as a descriptive statistic of changes in evenness over time. Our findings showed that bacterial community structure reflected the impact and the recovery from an anthropogenic disturbance. In addition, the intensity of impact and the rate of recovery to pre-perturbation structure were dose-dependent. These properties of bacterial community structures may potentially provide a metric for environmental assessment and regulation.

A linear model method for biodiversity-ecosystem functioning experiments.

Experiments that manipulate species richness and measure ecosystem functioning attempt to separate the effects of species richness (the number of species) from those of species identity. We introduce an experimental design that ensures that each species is selected the same number of times at each level of species richness. In combination with a linear model analysis, this approach is able to unambiguously partition the variance due to different species identities and the variance due to nonlinear species richness, a proxy measure for interactions among species. Our design and analysis provide several advantages over methods that are currently used. First, the linear model method has the potential to directly estimate the role of various ecological mechanisms (e.g., competition, facilitation) rather than the consequences of those mechanisms (e.g., the "complementarity effect"). Second, unlike other methods that are currently used, this one is able to estimate the impact of diversity when the contribution of individual species in a mixture is unknown.

Bacterial community diversity in cultures derived from healthy and inflamed ileal pouches after restorative proctocolectomy.

BACKGROUND: Pouchitis is believed to occur as a reaction to dysbiosis. In this study we assessed differences between mucosal bacterial communities cultured from noninflamed and inflamed ileal pouches. METHODS: Thirty-two ileal pouch patients, 22 with ulcerative colitis (UC) and 10 with familial adenomatous polyposis (FAP), underwent symptomatic, endoscopic, and histological assessment. The Objective Pouchitis Score (OPS) and the Pouch Disease Activity Index (PDAI) were used to diagnose pouchitis. Seven UC patients had pouchitis (UC+), 15 had a noninflamed pouch (UC-), 9 had a noninflamed pouch (FAP-), and 1 FAP patient had pouchitis (FAP+). Biopsies taken from the ileal mucosa of the pouch were cultured under aerobic and anaerobic conditions. Following standardized DNA extraction a polymerase chain reaction (PCR) was performed to generate 16S rRNA gene products. A "fingerprint" of the bacterial community within each sample was created using terminal-restriction fragment length polymorphism (T-RFLP) profiling. Species richness and evenness were determined using T-RF band lengths and relative band intensities. RESULTS: From the 64 DNA samples, 834 bands were detected, of which 179 represented different species (operational taxonomic units [OTUs]). The average species richness for the FAP-, FAP+, UC-, and UC+ groups was 26, 35, 23.9, and 29.6 per patient, with the average species diversity within the groups of 10.6, 29, 8.3, and 11.4, respectively. Similar trends were observed when the anaerobic and aerobic-derived bacterial groups were analyzed separately. CONCLUSIONS: No significant differences were found between the bacterial cultures derived from any of the clinical groups or between pouchitis and nonpouchitis patients.

Heterogeneous selection in a spatially structured environment affects fitness tradeoffs of plasmid carriage in pseudomonads.

Environmental conditions under which fitness tradeoffs of plasmid carriage are balanced to facilitate plasmid persistence remain elusive. Periodic selection for plasmid-encoded traits due to the spatial and temporal variation typical in most natural environments (such as soil particles, plant leaf and root surfaces, gut linings, and the skin) may play a role. However, quantification of selection pressures and their effects is difficult at a scale relevant to the bacterium in situ. The present work describes a novel experimental system for such fine-scale quantification, with conditions designed to mimic the mosaic of spatially variable selection pressures present in natural surface environments. The effects of uniform and spatially heterogeneous mercuric chloride (HgCl(2)) on the dynamics of a model community of plasmid-carrying, mercury-resistant (Hg(r)) and plasmid-free, mercury-sensitive (Hg(s)) pseudomonads were compared. Hg resulted in an increase in the surface area occupied by, and therefore an increase in the fitness of, Hg(r) bacteria relative to Hg(s) bacteria. Uniform and heterogeneous Hg distributions were demonstrated to result in different community structures by epifluorescence microscopy, with heterogeneous Hg producing spatially variable selection landscapes. The effects of heterogeneous Hg were only apparent at scales of a few hundred micrometers, emphasizing the importance of using appropriate analysis methods to detect effects of environmental heterogeneity on community dynamics. Heterogeneous Hg resulted in negative frequency-dependent selection for Hg(r) cells, suggesting that sporadic selection may facilitate the discontinuous distribution of plasmids through host populations in complex, structured environments.

Temporal scaling of bacterial taxa is influenced by both stochastic and deterministic ecological factors.

Microorganisms operate at a range of spatial and temporal scales acting as key drivers of ecosystem properties. Therefore, many key questions in microbial ecology require the consideration of both spatial and temporal scales. Spatial scaling, in particular the species-area relationship (SAR), has a long history in ecology and has recently been addressed in microbial ecology. However, the temporal analogue of the SAR, the species-time relationship, has received far less attention even in the science of general ecology. Here we focus upon the role of temporal scaling in microbial ecological patterns by coupling molecular characterization of bacterial communities in discrete island (bioreactor) systems with a macroecological approach. Our findings showed that the temporal scaling exponent (slope), and therefore taxa turnover of the bacterial taxa-time relationship decreased as selective pressure (industrial wastewater concentration) increased. Also, as the concentration of industrial wastewater increased across the bioreactors, we observed a gradual switch from stochastic community assembly to more deterministic (niche)-based considerations. The identification of broad-scale statistical patterns is particularly relevant to microbial ecology, as it is frequently difficult to identify individual species or their functions. In this study, we identify wide-reaching statistical patterns of diversity and show that they are shaped by the prevalent underlying ecological factors.

Frequency-dependent advantages of plasmid carriage by Pseudomonas in homogeneous and spatially structured environments.

The conditions promoting the persistence of a plasmid carrying a trait that may be mutually beneficial to other cells in its vicinity were studied in structured and unstructured environments. A large plasmid encoding mercury resistance in Pseudomonas fluorescens was used, and the mercury concentration allowing invasion from rare for both plasmid-bearing and plasmid-free cells was determined for different initial inoculum densities in batch-culture structured (filter surface) and unstructured (mixed broth) environments. A range of mercury concentrations were found where both cell types could coexist, the regions being relatively similar in the two types of environment although density-dependent in the unstructured environment. The coexistence is explained in terms of frequency-dependent selection of the mutually beneficial mercury resistance trait, and the dynamics of bacterial growth under batch culture conditions. However, the region of coexistence was complicated by conjugation which increased plasmid spread in the mixed broth culture but not the structured environment.