• Antibiotic Resistance in the Gut Microbiota

      Fouhy, Fiona; Teagasc Walsh Fellowship Programme; Irish Research Council (2014)
      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.
    • Composition of the early intestinal microbiota: Knowledge, knowledge gaps and the use of high-throughput sequencing to address these gaps

      Fouhy, Fiona; Ross, R Paul; Fitzgerald, Gerald F; STANTON, CATHERINE; Cotter, Paul D.; Irish Research Council for Science, Engineering and Technology; Teagasc Walsh Fellowship Programme; Science Foundation Ireland; 11/PI/1137 (Landes Bioscience, 01/05/2012)
      The colonization, development and maturation of the newborn gastrointestinal tract that begins immediately at birth and continues for two years, is modulated by numerous factors including mode of delivery, feeding regime, maternal diet/weight, probiotic and prebiotic use and antibiotic exposure pre-, peri- and post-natally. While in the past, culture-based approaches were used to assess the impact of these factors on the gut microbiota, these have now largely been replaced by culture-independent DNA-based approaches and most recently, high-throughput sequencing-based forms thereof. The aim of this review is to summarize recent research into the modulatory factors that impact on the acquisition and development of the infant gut microbiota, to outline the knowledge recently gained through the use of culture-independent techniques and, in particular, highlight advances in high-throughput sequencing and how these technologies have, and will continue to, fill gaps in our knowledge with respect to the human intestinal microbiota.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 2012205 (Biomed Central, 17/11/2015)
      Background The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.
    • High-Throughput Sequencing Reveals the Incomplete, Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic Treatment with Ampicillin and Gentamicin

      Fouhy, Fiona; Guinane, Caitriona M.; Hussey, Seamus; Wall, Rebecca; Ryan, C. Anthony; Dempsey, Eugene; Murphy, Brendan; Ross, R Paul; Fitzgerald, Gerald F; STANTON, CATHERINE; et al. (American Society for Microbiology, 04/09/2012)
      The infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA and rpoB-specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions of Proteobacteria (P = 0.0049) and significantly lower proportions of Actinobacteria (P = 0.00001) (and the associated genus Bifidobacterium [P = 0.0132]) as well as the genus Lactobacillus (P = 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, the Proteobacteria levels remained significantly higher in the treated infants (P = 0.0049), but the Actinobacteria, Bifidobacterium, and Lactobacillus levels had recovered and were similar to those in the control samples. Despite this recovery of total Bifidobacterium numbers, rpoB-targeted pyrosequencing revealed that the number of different Bifidobacterium species present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.