Membrane Fouling in Constant Permeate Flux Cross-Flow Microfiltration of Biological Solutions

This thesis investigates the fouling of a microfiltration membrane by biological solutions. Membrane fouling is recognized as a major drawback for the application of microfiltration in the purification of biotechnology products. Membrane fouling was analyzed and compared for filtrations performed with a hollow fiber microfiltration module operated at constant permeate flux using bovine serum albumin (BSA) solutions or Chinese hamster ovary (CHO) cell culture broths as feed solutions. A mechanistic model was developed to represent the fouling of a cross-flow microfiltration membrane operated at constant permeate flux. Fouling was observed as an increase in the transmembrane pressure (TMP) and assumed to occur first by pore blockage followed by cake formation over the blocked pores. The effect of the cross-flow action was described by the removal of deposits from the membrane surface thereby reducing the pore blockage and the mass of the cake. The model was fitted to the TMP profiles obtained during the filtration of BSA solutions and Chinese hamster ovary (CHO) cell culture broths with a 0.45 µm polysulfone hollow fiber membrane. According to the fitted model, pores blocked faster and more cake was formed with increasing BSA concentration. In the case of CHO cell culture broth, increasing the wall shear rate (proportional to cross-flow velocity) seemed to lead to the formation of a less pronounced cake but more pore blockage. The cross-flow mechanistic model was applied to the microfiltration of CHO cell culture supernatants harvested at different days of the fermentation process. The filtrations were performed at two different shear rates and with two different membrane pore sizes. The cell culture supernatant caused membrane fouling observed as an increase in both the TMP and the membrane hydraulic resistance estimated from water flux measurements at the end of the filtrations. The highest TMP increase was observed for the filtrations with the smaller membrane pore size (0.2 µm) and the higher shear rate (8000 s-1). The hydraulic resistance estimates of the fouled membrane also revealed a higher irreversible fouling for the smaller (0.2 µm) membrane pore size while the model analysis indicates that more fouling occurred at the entrance of the membrane pores. The shear rate was found to strongly influence the contribution of the reversible fouling to the total hydraulic resistance of the membrane. The cross-flow mechanistic model indicates a higher pore blockage for the most severe membrane fouling observed experimentally for the smaller membrane pore size and the higher shear rate. At the same time, a smaller cake deposit was predicted for the higher shear rate. The different cell culture harvest time investigated in this study did not reveal any differences in membrane fouling. BSA solutions were used to evaluate the contribution of BSA aggregates, fresh cell culture medium and the non-ionic surfactant Pluronic F-68 to membrane fouling. A simple empirical model was developed to represent the TMP increase and to derive the initial fouling rate. The initial fouling rate, the normalized TMP and the irreversible membrane resistance at the end of the filtration were analyzed to determine the effect of BSA solution characteristics on membrane fouling. The initial fouling rate increased with increasing BSA aggregate content consistent with a two-step fouling mechanism that was proposed for membrane fouling by BSA. Increased BSA concentration and the use of fresh cell culture medium compared to potassium phosphate buffer resulted in an increase in initial fouling rate, TMP and irreversible membrane fouling. The addition of the non-ionic surfactant Pluronic-F68 to the BSA solutions decreased the long-term fouling and the irreversible fouling but did not affect the initial fouling rate

Bacterial communities vary between sinuses in chronic rhinosinusitis patients

Chronic rhinosinusitis (CRS) is a common and potentially debilitating disease characterized by inflammation of the sinus mucosa for longer than 12 weeks. Bacterial colonization of the sinuses and its role in the pathogenesis of this disease is an ongoing area of research. Recent advances in culture-independent molecular techniques for bacterial identification have the potential to provide a more accurate and complete assessment of the sinus microbiome, however there is little concordance in results between studies, possibly due to differences in the sampling location and techniques. This study aimed to determine whether the microbial communities from one sinus could be considered representative of all sinuses, and examine differences between two commonly used methods for sample collection, swabs, and tissue biopsies. High-throughput DNA sequencing of the bacterial 16S rRNA gene was applied to both swab and tissue samples from multiple sinuses of 19 patients undergoing surgery for treatment of CRS. Results from swabs and tissue biopsies showed a high degree of similarity, indicating that swabbing is sufficient to recover the microbial community from the sinuses. Microbial communities from different sinuses within individual patients differed to varying degrees, demonstrating that it is possible for distinct microbiomes to exist simultaneously in different sinuses of the same patient. The sequencing results correlated well with culture-based pathogen identification conducted in parallel, although the culturing missed many species detected by sequencing. This finding has implications for future research into the sinus microbiome, which should take this heterogeneity into account by sampling patients from more than one sinus

Impact of Enhanced Staphylococcus DNA Extraction on Microbial Community Measures in Cystic Fibrosis Sputum

Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF

The combination of colistin and doripenem is synergistic against Klebsiella pneumoniae at multiple inocula and suppresses colistin resistance in an in vitro pharmacokinetic/pharmacodynamic model

There has been a resurgence of interest in aerosolization of antibiotics for treatment of patients with severe pneumonia caused by multidrug-resistant pathogens. A combination formulation of amikacin-fosfomycin is currently undergoing clinical testing although the exposure-response relationships of these drugs have not been fully characterized. The aim of this study was to describe the individual and combined antibacterial effects of simulated epithelial lining fluid exposures of aerosolized amikacin and fosfomycin against resistant clinical isolates of Pseudomonas aeruginosa (MICs of 16 mg/liter and 64 mg/liter) and Klebsiella pneumoniae (MICs of 2 mg/liter and 64 mg/liter) using a dynamic hollow-fiber infection model over 7 days. Targeted peak concentrations of 300 mg/liter amikacin and/or 1,200 mg/liter fosfomycin as a 12-hourly dosing regimens were used. Quantitative cultures were performed to describe changes in concentrations of the total and resistant bacterial populations. The targeted starting inoculum was 10(8) CFU/ml for both strains. We observed that neither amikacin nor fosfomycin monotherapy was bactericidal against P. aeruginosa while both were associated with rapid amplification of resistant P. aeruginosa strains (about 10(8) to 10(9) CFU/ml within 24 to 48 h). For K. pneumoniae, amikacin but not fosfomycin was bactericidal. When both drugs were combined, a rapid killing was observed for P. aeruginosa and K. pneumoniae (6-log kill within 24 h). Furthermore, the combination of amikacin and fosfomycin effectively suppressed growth of resistant strains of P. aeruginosa and K. pneumoniae In conclusion, the combination of amikacin and fosfomycin was effective at maximizing bacterial killing and suppressing emergence of resistance against these clinical isolates

Intravenous antibiotics for pulmonary exacerbations in people with cystic fibrosis

BACKGROUND: Cystic fibrosis is a multi-system disease characterised by the production of thick secretions causing recurrent pulmonary infection, often with unusual bacteria. Intravenous antibiotics are commonly used in the treatment of acute deteriorations in symptoms (pulmonary exacerbations); however, recently the assumption that exacerbations are due to increases in bacterial burden has been questioned. OBJECTIVES: To establish if intravenous antibiotics for the treatment of pulmonary exacerbations in people with cystic fibrosis improve short- and long-term clinical outcomes. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles and reviews and ongoing trials registers.Date of last search of Cochrane trials register: 27 July 2015. SELECTION CRITERIA: Randomised controlled trials and the first treatment cycle of cross-over studies comparing intravenous antibiotics (given alone or in an antibiotic combination) with placebo, inhaled or oral antibiotics for people with cystic fibrosis experiencing a pulmonary exacerbation. DATA COLLECTION AND ANALYSIS: The authors assessed studies for eligibility and risk of bias and extracted data. MAIN RESULTS: We included 40 studies involving 1717 participants. The quality of the included studies was largely poor and, with a few exceptions, these comprised of mainly small, inadequately reported studies.When comparing treatment with a single antibiotic to a combined antibiotic regimen, those participants receiving a combination of antibiotics experienced a greater improvement in lung function when considered as a whole group across a number of different measurements of lung function, but with very low quality evidence. When limited to the four placebo-controlled studies (n = 214), no difference was observed, again with very low quality evidence. With regard to the review's remaining primary outcomes, there was no effect upon time to next exacerbation and no studies in any comparison reported on quality of life. There were no effects on the secondary outcomes weight or adverse effects. When comparing specific antibiotic combinations there were no significant differences between groups on any measure. In the comparisons between intravenous and nebulised antibiotic or oral antibiotic (low quality evidence), there were no significant differences between groups on any measure. No studies in any comparison reported on quality of life. AUTHORS' CONCLUSIONS: The quality of evidence comparing intravenous antibiotics with placebo is poor. No specific antibiotic combination can be considered to be superior to any other, and neither is there evidence showing that the intravenous route is superior to the inhaled or oral routes. There remains a need to understand host-bacteria interactions and in particular to understand why many people fail to fully respond to treatment

Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention

© 2016 International Society for Microbial Ecology All rights reserved. Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors