Proteomics as the final step in the functional metagenomics study of antimicrobial resistance

peer-reviewedThe majority of clinically applied antimicrobial agents are derived from natural products generated by soil microorganisms and therefore resistance is likely to be ubiquitous in such environments. This is supported by the fact that numerous clinically important resistance mechanisms are encoded within the genomes of such bacteria. Advances in genomic sequencing have enabled the in silico identification of putative resistance genes present in these microorganisms. However, it is not sufficient to rely on the identification of putative resistance genes, we must also determine if the resultant proteins confer a resistant phenotype. This will require an analysis pipeline that extends from the extraction of environmental DNA, to the identification and analysis of potential resistance genes and their resultant proteins and phenotypes. This review focuses on the application of functional metagenomics and proteomics to study antimicrobial resistance in diverse environments.The Alimentary Pharmabiotic Centre is a research centre funded by Science Foundation Ireland (SFI). This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273 and by FP7 funded CFMATTERS (Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerba- tions: Results Stratified, Grant Agreement no. 603038)

A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and ß-lactam resistance genes in the gut microbiota

peer-reviewedBackground: The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes. Results: The strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and β-lactam (including bla OXA, bla TEM, bla SHV and bla CTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes. Conclusions: This study has demonstrated that the gut can be a significant source of aminoglycoside and β-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes.Fiona Fouhy is in receipt of an Irish Research Council EMBARK scholarship and is a Teagasc Walsh fellow. Research in the PDC laboratory is also supported by the Irish Government under the National Development Plan through the Science Foundation Ireland Investigator award 11/PI/113

16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform

peer-reviewedBackground Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Numbers SFI/12/RC/2273 and 11/PI/1137 and by FP7 funded CFMATTERS (Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified, Grant Agreement no. 603038)

Manufacturing single use systems with quality in mind: How to assure performance, robustness, and sterility of single use systems

Quality control during the manufacturing of single use systems is critical. With traditional stainless-steel systems, the end user has significant control over the design, construction, qualification, validation, and maintenance of the system. When implementing a single-use system, the supplier of the single use product takes responsibility for many of these functions from the user. It is therefore important that the single use supplier has established and uses a robust quality control system. This presentation will highlight the quality systems, processes, facilities, and personnel required to assure the performance, robustness, and sterility of single use systems. The following topics will be covered: Single-use assembly validation Qualification of components Sterilization qualification Manufacturing processes Quality control Release testing Certification Risk mitigation practices Process particulate control Operator training Leachables & Extractables Patient safety evaluation, study design Support by the supplie

Sterile media hold scale-up using MOBIUS® single-use technology

The benefits of single-use systems (SUS) in biopharmaceutical manufacturing are well understood, and their use is widespread in the manufacture of mAb, rProteins, and related therapies. New frontiers in medicine such as cell and gene therapy present an opportunity for SUS to enable speed to the clinic, however some unique hurdles must be overcome. This poster outlines the collaborative development of a single-use process to address the challenge of supplying sterile media to a bioreactor for inoculation and growth of human tissue cells. To alleviate time constraints, cleaning concerns, and contamination risks, the biopharmaceutical manufacturer chose to employ single-use technology when conducting a 5-fold scale up from a glass bottle process. A significant challenge with this human tissue cell culture process is the 60-day sterile media hold at the cell culture temperature of 36oC, during which time the bioreactor is intermittently perfused with fresh media. The Mobius® Mix50 single-use mixer (SUM) solves this challenge by first beginning with a sterile, gamma-irradiated mixer bag to eliminate concerns over validation of CIP and SIP cycles. Next, a low-pressure overlay is maintained with a carefully-sized hydrophobic vent filter, to prevent contaminants from entering the sealed mixer container. Process variables requiring assessment for this application include the air overlay pressure and flow rate, the liquid (media) flow rate during filling and draining of the SUM, sizing of the vent filter area, the liquid volume in the SUM, and the sterile condensate collection rate. A series of experiments provided a repeatable and scalable single-use solution for implementation into the manufacturing process. This novel application demonstrates the flexibility of single-use in the rapidly expanding clinical market of products derived from human cells with the unique challenges they present

Antibiotic resistance in the gut microbiota

Antibiotic resistance is an increasing threat to our ability to treat infectious diseases. Thus, understanding the effects of antibiotics on the gut microbiota, as well as the potential for such populations to act as a reservoir for resistance genes, is imperative. This thesis set out to investigate the gut microbiota of antibiotic treated infants compared to untreated controls using high-throughput DNA sequencing. The results demonstrated the significant effects of antibiotic treatment, resulting in increased proportions of Proteobacteria and decreased proportions of Bifidobacterium. The species diversity of bifidobacteria was also reduced. This thesis also highlights the ability of the human gut microbiota to act as an antibiotic resistance reservoir. Using metagenomic DNA extracted from faecal samples from adult males, PCR was employed to demonstrate the prevalence and diversity of aminoglycoside and β-lactam resistance genes in the adult gut microbiota and highlighted the merits of the approach adopted. Using infant faecal samples, we constructed and screened a second fosmid metagenomic bank for the same families of resistance genes and demonstrated that the infant gut microbiota is also a reservoir for resistance genes. Using in silico analysis we highlighted the existence of putative aminoglycoside and β-lactam resistance determinants within the genomes of Bifidobacterium species. In the case of the β- lactamases, these appear to be mis-annotated. However, through homologous recombination-mediated insertional inactivation, we have demonstrated that the putative aminoglycoside resistance proteins do contribute to resistance. In additional studies, we investigated the effects of short bowel syndrome on infant gut microbiota, the immune system and bile acid metabolism. We also sequenced the microbiota of the human vermiform appendix, highlighting its complexity. Finally, this thesis demonstrated the strain specific nature of 2 different probiotic CLA-producing Bifidobacterium breve on the murine gut microbiota

The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

peer-reviewedBackground High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed. Methods Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed. Results No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples. Conclusions Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.Jennifer Deane is in receipt of a Teagasc Walsh Fellowship. The authors and their work were supported by the Science Foundation Ireland and funded by the Centre for Science, Engineering and Technology (SFI-CSET) grant 02/CE/B124 and by FP7 funded CFMATTERS (Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified, Grant Agreement no. 603038). The Alimentary Pharmabiotic Centre is a research centre funded by Science Foundation Ireland (SFI). This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

peer-reviewedBifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.Fiona Fouhy is in receipt of an Irish Research Council for Science, Engineering and Technology EMBARK scholarship and is a Teagasc Walsh fellow. Research in the PDC laboratory is supported by the Irish Government under the National Development Plan through the Science Foundation Ireland Investigator award 11/PI/1137. Research in the RPR, CS, PDC and DvS laboratories is also supported by the Science Foundation of Ireland-funded Centre for Science, Engineering and Technology, the Alimentary Pharmabiotic Centre (grant no.s 02/CE/B124 and 07/CE/B1368) and a HRB postdoctoral fellowship (Grant no. PDTM/20011/9) awarded to MOCM

Rousseau: his social ideas and their influence ..

This item has been digitized by the Internet Archive. Typewritten sheets in cover. Thesis (M.A.)--Boston University Bibliography: 5 p. at end

Mapping research across the undergraduate curriculum in UCC

UCC identifies itself as a research-led University and has stated the ambition to strengthen the integration of research, teaching and learning by maximising opportunities for students to participate in research programmes throughout their undergraduate studies. The number of undergraduate programmes with student-involved research from first year onwards is an important measure of this ambition. A curriculum analytics project was enacted by CIRTL staff and Academic Systems Administration to gather evidence of research-oriented and research-based teaching in undergraduate programmes offered to students via the CAO system in 2015/2016. The review showed that 55% of undergraduate programmes make explicit mention of research and inquiry in their programme learning outcomes. Analysis of module learning outcomes further showed that 45% of the reviewed programmes provide students with exposure to research-based or research-oriented teaching across the duration of their programme. The project provides an important baseline of existing research in the undergraduate curriculum, it uncovers exemplar activities across a range of subject areas and disciplines, and extends the vocabulary around research and inquiry to include discipline-specific approaches and understandings. Future work will include gathering feedback from staff and qualitative research with students to correct any inaccuracies in the data with a view to refining the search query and running a regular, more automated analysis