APC Microbiome Ireland - Doctoral Theses

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Identification of bacteria-regulated mechanisms for the pathogenesis of inflammatory bowel diseases (IBD) and development of upconverting nanoparticle (UCNP) luminescence imaging for the monitoring of gut bacteria
(University College Cork, 2022) Singh, Raminder; Melgar Villeda, Silvia; Andersson-Engels, Stefan; Shanahan, Fergus; Science Foundation Ireland; APC Microbiome Institute
Inflammatory bowel disease (IBD) is a chronic inflammatory state of the gastrointestinal tract, including two inflammatory conditions Crohn’s disease (CD) and ulcerative colitis (UC). The environment, the gut microbiota, the genetic make-up and the immune response are believed to contribute to the aetiology of the disease. Non-steroidal anti-inflammatory drugs (NSAIDs) are believed to exacerbate inflammation in patients with IBD. However, the literature demonstrates no consensus on the association between NSAID use and IBD, with some studies reporting that only high dose NSAID treatment are more likely for IBD exacerbation or relapse. In this study, using the piroxicam-accelerated model of colitis in interlukin-10 deficient (IL-10-/-) mice, we showed that mice fed 100 ppm piroxicam in food for 9 days (high dose) followed by 5 days of regular chow, develop colitis. The colitic phenotype was associated with activation of caspase-8, NLRP3 inflammasome and apoptosis and independent of initial gut microbiota. However, 5 days exposure (low dose) to 100 ppm piroxicam did not lead to colitis development. Adherent-invasive Escherichia coli (AIEC) is widely prevalent and heterogeneously present in the mucosa of IBD patients, particularly in Crohn’s disease. We hypothesise that the presence of AIEC in the gut of IBD patients might explain the NSAID-induced inflammation in IBD under low dose treatment (5 days piroxicam treatment). Indeed, 5 days piroxicam exposure was sufficient to induce colitis in AIEC-precolonised animals, indicating a synergism between AIEC colonisation and piroxicam treatment. Inhibition of NLR family pyrin domain containing 3 (NLRP3) or Caspase-8 activity ameliorated colitis. This synergism was lost under high dose piroxicam (9 days exposure) treatment. Our data indicate that under low dose NSAID treatment, AIEC can potentiate NSAID-induced inflammation in IL-10-/- mice by regulating the intestinal epithelial function and the immune response, highlighting its potential role in NSAID-induced inflammation in IBD patients exposed to low dose NSAIDs. Although, under high dose, NSAID itself is sufficient to induce colitis. This data suggests that the lack of consistency in the association between NSAID use and IBD could be explained by the NSAID dose and the presence of AIEC in the patients with IBD. Future studies should consider both these factors while studying association between NSAID use and symptomatic worsening in IBD. The role of microbes in IBD is supported by various studies performed in animal models where germ free mice are protected from intestinal inflammation. However, the precise interaction(s) between microbes and the host are not well understood. In vivo imaging techniques using custom designed fluorescent probes and bioluminescence have been classically used to track gut microbes. Several disadvantages while using fluorescent probes include autofluorescence, photobleaching and photodamage. To overcome these limitations, we propose to develop upconverting nanoparticles (UCNPs) luminescence imaging for monitoring of gut bacteria. Their unique property of photon upconversion enables them to convert low energy near-infrared (NIR) light into higher energy visible/NIR light offering greater tissue penetrance and signal-to-noise ratio. Here, we investigated the possibility of using UCNP 1) to image a single gut bacterium (e.g., AIEC) using UCNP-conjugated anti-E. coli antibody, and 2) to image the endogenous gut bacteria using metabolic labelling of bacterial peptidoglycan with azido-D-amino acids and strained-cyclooctynes such as Dibenzocyclooctyne (DBCO) -functionalised UCNPs. We showed a proof of principle for both these approaches using a fluorescent dye, but we were not able to replicate it with UCNP. Preliminary data suggests that the size difference between the UCNPs and the fluorescent dyes may be one of the potential reasons for lack of labelling. Future experiments should consider using smaller UCNPs and/or use a polyethylene glycol (PEG) linker between DBCO and UCNPs to increase the length of DBCO-UCNP construct and reduce the steric hinderance for better stability of UCNP-bacteria conjugation.
The use of lactic acid bacteria as methane mitigation strategies in ruminants
(University College Cork, 2023) Doyle, Natasha; Stanton, Catherine; Ross, R. Paul; Hill, Colin; Horizon 2020
Lactic acid bacteria (LAB) have often been cited as potential strategies for the reduction of methane emissions from ruminants, however more research is needed to identify whether LAB can be an effective methane mitigation option in ruminant livestock. The aim of this thesis was to investigate the potential of LAB as methane mitigation strategies in ruminants, by determining the effect of LAB on methane production, animal health and productivity. Chapter 1, a literature review, addresses the topic of greenhouse gas (GHG) emissions in agriculture, including methane, nitrous oxide and carbon dioxide. Methane production from ruminants is influenced by a wide variety of factors, including feed additives such as lactic acid-utilizing bacteria, LAB and yeast. This chapter focuses on the use of direct fed microbials (DFMs) for the reduction of GHG emissions in agriculture. The strengths and challenges associated with each strategy are also explored. Chapter 2 focuses on the use of LAB specifically for methane mitigation in ruminants. Enteric fermentation is the largest anthropogenic source of agricultural methane and, as such, requires innovative solutions for the reduction of methane from livestock. This chapter reviews current literature and provides a comprehensive analysis on the potential use of LAB as methane mitigation strategies. To determine the methane mitigation potential of LAB, several species of methanogens (methane producing microorganisms) were cultivated. In Chapter 3, a total of 6 methanogenic species of both human and ruminant origin were cultivated. Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii were chosen for cultivation as they comprise ~75% of the archaeal population within the rumen. Several human methanogens were also chosen for cultivation to allow for comparison of cultivation techniques required for these methane producing microorganisms and for the determination of methane production from several methanogenic strains. Gas chromatography methods determined that methanogens of rumen origin produced almost 4000 times more methane (90g/L) in vitro, when compared to human methanogen strains (0.025g/L). Establishment of methanogen cultivation techniques were essential to allow for further research to be carried out in Chapter 4. Methane inhibition trials assessed the potential of LAB and various bacterial families, including Staphylococcus, for the reduction of methane in vitro. Staphylococcus capitis APC 2918 resulted in the best methane inhibition of 54% and 69% in Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii, respectively. Commercially available LAB strains (provided by a commercial partner), Lactobacillus plantarum LP58 and Lactococcus lactis subsp. lactis SL242, demonstrated methane reduction up to 28% against Mbb. gottschalkii (P <0.05). The screening process reported in chapter 4 resulted in a biobank of 20 strains with bacteriocin capabilities and possible anti-methanogenic potential. Both of the commercially available LAB strains, Lactobacillus plantarum LP58 and Lactococcus lactis subsp. lactis SL242, with demonstrated methane reduction effects of 9-28% in vitro, were chosen as silage co-inoculants for use in an animal intervention study. Chapter 5 determined the effects of these LAB co-inoculants on silage quality. LAB when administered as co-inoculants resulted in good quality silage in terms of pH (4.27, SD 0.653), dry matter percentages (26%, SD 1.532) and overall appearance. However, when compared to control silage with no silage inoculant, little statistical difference was seen. This LAB inoculated silage was subsequently used in a 7 week animal intervention study in Chapter 6. Animal intervention studies resulted in reduced methane emissions of 5.7% (P < 0.01) in 30 late-lactating Holstein dairy cattle. Ruminal content of cattle fed LAB inoculants resulted in a 2-log reduction of Proteobacteria (P = 0.006), a major phylum of gram-negative bacteria. Cyanobacteria were also significantly reduced by ~3 logs in treatment cattle (P = 0.007). Cattle treated with LAB inoculated silage produced milk with high lactose content (4.6%, P<0.05), reduced milk urea nitrogen between weeks 2 and 5 of the animal intervention (P< 0.05), and greater free fatty acids (9.92%, P <0.05). Taken together, the results of this thesis demonstrate that LAB provide an effective solution for the reduction of methane emissions in ruminants, while simultaneously improving animal health and maintaining productivity. Through the knowledge gathered, it can be said that LAB have the potential to provide methane mitigation solutions which will aid in the development of a competitive and sustainable Agri-food sector.
Impact of antibiotics on the gut and milk microbiome
(University College Cork, 2023) Patangia, Dhrati; Stanton, Catherine; Ross, R. Paul; Science Foundation Ireland
While antibiotics are lifesaving, they come at the cost of antimicrobial resistance (AMR) and collateral damage to microbiome. Using single-sample and longitudinal study designs, this thesis aimed to study antibiotic resistance genes (ARGs) spread using shotgun metagenomics sequencing. ARGs developed in animals can enter humans through the food chain. We examined the effect of single antibiotic use on microbiome and resistome in milk from dairy cows longitudinally. The microbial diversity increased from colostrum to later time-points in the antibiotic groups which was not prominent in the no-ab group. Further, microbial composition in all groups was different, leading to distinct clustering of no- antibiotic from antibiotic groups. All groups had a wide resistome profile, with antibiotic groups showing higher ARG abundance. Also, high abundance of mastitic pathogens was absent in the no-ab group. The results show that prophylactic antibiotics during DCT is not essential. We next longitudinally studied microbiome and resistome in infants divided into three groups [CSab (C-section/antibiotic), CSnoab (C-section/antibiotic naive) and VDnoab (Vaginal delivered/antibiotic naive)] based on delivery mode and antibiotic use during early life. CSab group showed low initial microbial diversity, which increased gradually and slowly. CSab group demonstrated significant associations to antibiotic classes corresponding to the antibiotic administered to infants in this group. Taxa belonging to Gammaproteobacteria were dominant carriers of ARGs, most being non persisters. The results show that early antibiotic exposure can have immediate and long-term effects on the infant microbiome. Next, publicly available shotgun-sequencing data was downloaded, analysed, resulting in a catalogue of early-life gut genomes of infants below three years of age which was used to study the global resistome. Gram-negative bacteria such as Escherichia, Enterobacter, Citrobacter, Klebsiella had highest ARG abundance with Glycopeptides, fluoroquinolone, macrolides, tetracyclines being most abundant classes. High abundance of ARGs was positively related to the socioeconomic status and healthcare access index of a country. These results confirm infant gut microbiota as ARG reservoir. Lastly, the gut microbial and resistome profile of adult cystic fibrosis (CF) patients was studied due to their chronic antibiotic use. The CF group had a microbial composition significantly distinct from controls with higher abundance of ARGs and virulence factors. CF group showed strong association to antibiotic classes administered to individuals in this group. Our results demonstrate the need to investigate the resistome and functional profile in this patient group; as antibiotic overuse can lead to MDR, aggravating the health status. This thesis sheds light on the microbial and resistome profile in previously unexplored manners and provides a baseline for researchers and policy makers to design pre-emptive and proactive measures to maximise health restoration and minimise ARG development.
LAB as probiotics and silage inoculants for enteric methane inhibition in ruminants
(University College Cork, 2022-10-19) Mbandlwa, Philiswa; Stanton, Catherine; Ross, R. Paul; Hill, Colin; FACCE-ERAGAS
There is a constant need for antimicrobial agents and producers that can be used in animal feed and other applications in the agricultural industry. This need justifies the research on novel antimicrobial alternatives such as bacteriocins. Data showed the agricultural environment as a source of LAB, dominated by Lactobacillus species with bacteriocin-producing abilities. Subsequently, an investigation on using CFS and bacteriocins from LAB to reduce methane production in Methanobrevibacter found that L. plantarum (LP58) was most effective in reducing methane from M. gottschalkii while individual strains of L. lactis affected methane production in Methanobrevibacter differently. Purified bacteriocins nukacin and lacticin 3147 did not influence in vitro methane production. The nutritive and fermentative properties of L. plantarum and L. lactis were detailed as silage co-inoculants and these inoculants enhanced the beneficial microbes in the silage and resulted in silage with significantly higher lactic acid concentration. Finally, an animal feeding trial showed significant methane reduction in the dairy cattle fed LAB-treated silage. Gaining insight into the different ruminotypes can enable more targeted approaches for methane mitigation. An investigation of the role of LAB in modifying the rumen microbiome and metabolome showed that LAB positively impacts the rumen by significantly increasing acetate and butyrate production while significantly decreasing Proteobacteria. Livestock farming is a significant sector in the Irish economy as it creates jobs, enables export, and is an intricate part of Irish culture. Although it will be challenging for Irish agriculture to meet its climate targets, concerted efforts, such as using LAB as enteric methane mitigators, can enable Ireland to reach its target of reducing atleast 10% of its GHG emissions by 2030. If overall methane emissions are lowered, the national dairy herd can be maintained or increased, and jobs can be created while continuing to supply meat and milk to the growing world population. Future research will focus on finding even more potent methane reducing LAB and characterisation and elucidating the mechanism of methane inhibition in vitro using LAB CFS.
Characterization of two prevalent yet distinct conjugation systems among lactococci
(University College Cork, 2022-12-08) Ortiz Charneco, Guillermo; van Sinderen, Douwe; Mahony, Jennifer; Science Foundation Ireland; DSM Food and Beverage
Lactic acid bacteria are Gram-positive, non-motile, lactic acid-producing microorganisms, with Lactococcus lactis and Lactococcus cremoris representing species that are extensively applied as starter bacteria in dairy fermentations. Starter culture providers continuously aim to expand their culture catalogues and enhance their existing cultures to maintain pace with the ever-changing consumer demand for products with diverse organoleptic attributes. Plasmids present in L. lactis and L. cremoris species harbor genes associated with many important technological traits, such as bacteriophage resistance, lactose metabolism and exopolysaccharide (EPS) production. There are practical limitations associated with the isolation of new strains, while there are regulatory hurdles associated with the application of genetically-modified organisms (GMOs). Consequently, the enhancement of existing starter strains through natural gene transfer methods including conjugation can be a highly advantageous approach, though with its challenges as (plasmid) conjugation efficiencies between lactococcal strains reported in literature have been very low. This thesis describes the optimization and characterization of two plasmid-encoded conjugation systems that are commonly found in L. lactis and L. cremoris strains. Chapter II details an optimized protocol for conjugation with the lactococcal conjugative plasmid pNP40, which represents a highly prevalent conjugation system among lactococci. A combined approach of in silico and mutational analyses facilitated the functional assignment of many conjugation-associated genes including a transcriptional repressor of this system. Furthermore, certain non-conjugative plasmids were observed to co-mobilise together with the conjugative plasmid. Chapter III details the mutational and functional analysis of the pUC11B conjugation gene cluster, which was demonstrated to be capable of self-transfer using the optimized conjugation protocol described in Chapter II. This conjugation system, along with those encoded by plasmids pMRC01 and pAF22, represents the second most prevalent type of conjugation system among lactococcal plasmids in public databases. Within the conjugation gene cluster, a transcriptional repressor and an anti-restriction modification (RM) system were identified. Interestingly, the anti-RM system promotes more efficient conjugation of this plasmid into recipient strains with Type II or Type III RM systems. Chapter IV focuses on the transcriptional analysis of the conjugation gene clusters of pNP40 and pUC11B, revealing the presence of three and five promoters, respectively, as well as their precise transcriptional start sites. Moreover, regulation of several of these promoters appears to be mediated by the individual or concerted action of the corresponding relaxase and transcriptional repressor associated with each conjugation system. In Chapter V, pNP40- and pUC11B-mediated mobilisation of smaller, non-conjugative plasmids present in the same donor strains is reported. The origin of transfer (oriT) sequences of pNP40 and pUC11B were determined. Sequences similar to these oriT sequences present in other, non-conjugatable plasmids had been reported to be required for their mobilisation. Moreover, the relaxases of both conjugation systems presented specific nickase activity to their respective oriT-containing sequences. Finally, the presence of additional copies of the auxiliary gene mobC in the same donor strain was shown to confer a high-frequency mobilisation phenotype. Chapter VI details the identification and characterization of surface adhesins encoded within the two prevalent lactococcal conjugation gene clusters. The surface adhesion functions of both pNP40 and pUC11B conjugation systems appear to be interchangeable between these two otherwise divergent systems. Additionally, overexpression of these proteins promoted significant cell clumping phenotypes, reminiscent of their cell adhesin function. In summary, the thesis provides a wide range of novel findings which facilitated comprehensive insights into the functionality, prevalence and diversity of lactococcal conjugation systems and the means by which conjugation is regulated. Understanding these systems and the enhancement of conjugation processes is invaluable to the rapid development of robust and technologically desirable starter cultures for sustainable food production systems.

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