- Volume 1, Issue 1A, 2019

As the consequences of increasing bacterial resistance to traditional antibiotics become evident, the importance of research and development of therapeutic alternatives is apparent. Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host. However, whether and how bacteriophages can kill intracellular bacteria in a human cell environment remains elusive. E. coli strains displaying the K1 polysaccharide capsule virulence factor, are nosocomial pathogens responsible for urinary tract infections (UTIs), neonatal meningitis and potentialprecursors for septicaemia. These different types of infections were modelled in vitro by infecting human bladder epithelial cells (T24 HTB-4) and humancerebral endothelial cells (hCMEC/D3) with E. coli EV36, a strain displaying the K1 capsule. The infected human cells then received in vitro phage therapy using bacteriophage K1F that specifically targets E. coli strains displaying the K1 capsule. The rate of bacterial infection and the molecular and cellular mechanisms of in vitro phage therapy was analysed by means of flow cytometry, confocal and live microscopy. We show that rfp-tagged E. coli EV36-RFP and gfp-tagged bacteriophage K1F-GFP, enter the human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP can efficiently kill intracellular E. coli EV36-RFP in human urinary bladder epithelial cells and humancerebral endothelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy. Collectively this data contribute evidence-based knowledge for the ongoing development of phage therapy.

Staphylococcus aureus is one of the dominant organisms isolated from the airways of cystic fibrosis (CF) patients, particularly early in life, and is usually regarded as pathogenic. However, there remains significant gaps in our understanding of the role of S. aureus in the progression of pulmonary infection and lung disease in CF. Mouse models of S. aureus lung infection, even in CF animals, frequently demonstrate pneumonia and abscesses of the lung, a phenomenon very rarely observed in people with CF. Furthermore, live host models are associated with high costs and are limited in duration and sample size for ethical reasons. Most in vitro models fail to consider the influence of host tissue interaction or spatial structure on the development and persistence of infection. We have previously described an ex vivo pig lung model (EVPL) of cystic fibrosis for Pseudomonas aeruginosa lung infection. Here we show the progression of this model to support the growth of Staphylococcus aureus. Our data suggests that, in our model, S. aureus cells may preferentially aggregate in artificial sputum rather than adhere to lung tissue. In the context of historical case reports, this result potentially reflects the clinical situation in cystic fibrosis more accurately than mouse models and could have substantial clinical significance.

Aging can be defined as an accumulation of damage, or a loss of function, with increasing age. For bacteria, it has generally been assumed that the mechanism used to cope with aging is the asymmetric segregation of damage at division, so that all of the damage is inherited by one cell and the other is therefore rejuvenated. Another, often neglected, mechanism is to repair the damage; our previous computational modelling work has found that an optimized, fixed rate of repair is fitter than damage segregation in well-mixed environments such as chemostats. The predominant mode of growth for bacteria is in biofilms, however, and here we investigate aging in biofilms using the individual-based model iDynoMiCS. In addition to the previously used damage segregation and fixed repair strategies, we introduced adaptive repair: sensing the current damage levels within the cell and responding to this by investing in damage repair machinery. We found that the optimal method for dealing with cellular damage varies with the environment being investigated. The investment of additional resources into adaptive repair is only beneficial when competition is sufficiently strong, in the chemostat and in biofilms, and the speed at which the fittest strategy becomes apparent depends upon the initial density of cells. When the bacterial cells are dense initially, and thus the competition between strategies is stronger, the adaptive repair strategy emerges as the winner much more rapidly.

Candida species are commensal yeasts but are also responsible of life-threatening infection in at-risk populations, like new-born or immunocompromised patients. Candida albicans is the most common causative species, and the most studied. Moreover, non-albicans Candida species as Candida glabrata, Candida parapsilosis and Candida tropicalis cause a large proportion of infections. In our study we investigated the interaction between four Candida species and human vaginal epithelial cells A431 by using a dual RNA-seq method. Our aim is to identify the different transcriptomic response of each yeast, and of the host, during the infection of human cells. Gene Ontology analysis of up-regulated genes in the yeasts during infection implicated the ergosterol (ERG) pathway in C. parapsilosis only. We therefore investigated the role of the ERG pathway in the three Candida species in which it is currently possible to generate gene knockouts. The transcriptional factor Upc2 is the main regulator of ERG gene expression in C. albicans and C. parapsilosis. C. glabrata has two UPC2 orthologs, called CgUPC2A and CgUPC2B. We found that deleting CgUPC2A or CgUPC2B or both together does not reduce the damage inflicted by C. glabrata on host cells. However, deleting UPC2in C. albicans greatly reduces damage. Deleting UPC2 in C. parapsilosis appears to reduce damage of host cells; however further investigation is required. Our results show the that the role of ergosterol pathway in the host pathogen interaction differs between Candida species.

MGB-BP-3 (MGB) is a novel synthetic antibiotic inspired by Distamycin – a natural product that is capable of binding to the minor groove of DNA. MGB has a high bactericidal activity against a broad range of Gram-positive bacteria without the toxicity associated with the natural products that it was inspired by. Its oral formulation, developedfor the treatment of Clostridium difficile infections, is currently progressing through a phase 2 clinical trial. This study investigatesthe mode of action of this novel antibiotic. To allow better understanding of MGB’s mode of action, RNA-Seq analysis was undertaken on S. aureus following challenge with 0.5 x MIC (0.1 µg ml−1) MGB-BP-3. Triplicate samples of RNA were extracted at 10 min after challenge. RNA-Seq analysis identified 698 transcripts showing significant changes in expression profile, which were confirmed by quantitative RT-PCR. Amongst these, 62 essential genes showed transcriptional arrest. Glycolysis, pentose phosphate pathway and the TCA cycle were affected. In addition, biosynthesis of nucleotides and certain amino acids were altered and Biolog phenotype arrays were performed in the presence of MGB to confirm this. DNA binding assays demonstrated MGB binding to intergenic regions upstream of strongly down-regulated essential genes (mraY and dnaD). Attempts to evolve resistance to MGB have so far been unsuccessful unlike with the rifampicin control. In conclusion our findings are consistent with a bactericidal mode of action of MGB at the transcriptional level of multiple essential genes.

The human gut microbiota is complex, dense, and hugely influential to health. Imbalances in the microbiota have been associated with numerous disease states, in many cases due to the overgrowth of Enterobacteriaceae, such as Escherichia coli. We describe an oligonucleotide antimicrobial that selectively reduces levels of Enterobacteriaceae in vitro, in a model of the human colonic microbiota, and in vivo in a murine study, whilst leaving the core microbiota intact. The antimicrobials are Transcription Factor Decoys (TFDs) that bind to and competitively inhibit an identified transcription factor necessary for growth in the intestine. This is highly conserved amongst Enterobacteriaceae and controls anaerobic respiration and response to nitrosative stress caused by the innate immune response of the host. A nanoparticulate formulation delivers the TFDs to the cytoplasm of E. coli, as visualized by confocal microscopy, and rapidly kills the bacteria under microaerobic conditions. When applied to anin vitro model of the human intestinal microbiota the TFD produced a decrease up to log10 6 c.f.u. ml−1 in coliforms within the Enterobacteriaceae family while other families remain intact. When delivered orally to the intestines of mice similar results were seen: Enterobacteriaceae were decreased or cleared from the wild-type intestinal microbiota while the remaining bacteria were unaffected. This demonstrates that TFDs can be used to make precise changes to the microbiota and has utility in testing associations between dysbiosis and disease and developing microbiota targeted therapeutics.

The long-term reproductive success of a lineage depends on its ability to tolerate a wide range of environmental conditions and for a virus a substantial part of its environment is the host that it infects. Viruses may overcome limitations of a given host environment by switching to other available hosts. Experiments addressing host switching may pave the way for an improved understanding of the emergence of new viral diseases. Here, a model bacteriophage, φX174, its canonical laboratory bacterial host strain, Escherichia coli, and a novel host, Salmonella Typhimurium were employed. Bacteriophage φX174 adaptation was studied at population level in a bioreactor. We analyzed phenotypes and genotypes arising during continuous evolution of φX174 on alternating hosts for four consecutive periods of 10 days each. The fitness and adsorption of each viral population were measured using qPCR in liquid culture. Deep sequencing analysis of isolates was carried out to determine the genetic basis of pleiotropic costs and to characterize allelic variations occurring during growth. Some alleles specific to one host were lost or reduced in frequency in the alternative host while other alleles (not present in the ancestor) were shared between hosts. The fitness effects of specific alleles were examined in isolation through targeted mutagenesis. This work contributes to a better understanding of some of the general constraints, costs and benefits influencing the evolution of parasite populations as they adapt to the complexities of a novel host environment- a key consideration during the emergence of infectious diseases.

Campylobacter spp. is a leading cause of foodborne illness globally. The pathogen colonises the gastrointestinal tract of the host, where small concentrations of neuroendocrine hormones are also secreted. Epinephrine and norepinephrine are neuroendocrine hormones involved in the stress response that have been shown to promote the expression of virulence factors in pathogens including E. coli, Salmonella spp., and Campylobacter spp. In our study Campylobacter jejuni strains from human infection and broiler farms that were supplemented with epinephrine and norepinephrine showed increased growth characterised by shorter lag phases and higher maximum OD595, and enhanced pathogenicity characterised by increased motility, attachment to and invasion of Caco-2 cells. The data obtained suggests that host stress may promote C. jejuni proliferation and pathogenicity.

Streptomyces is a genus of soil dwelling actinomycetes that play an important role in plant health through association with plant roots. They provide an array of benefits to the plant host such as infectious disease prevention and plant growth promotion. We investigate the role of nitric oxide (NO), a ubiquitous signalling molecule used by plants and bacteria alike, in root colonisation by Streptomyces coelicolor. Plating studies were conducted for Arabidopsis thaliana and Triticum aestivum. Relative colonisation was determined by comparing selective recovery of marked mutant strains, alongside a marked control. The effect of increased endogenous NO was interrogated with a deletion mutant of nsrR-hmpA – genes responsible for NO detoxification in Streptomyces. Strains were also engineered to express recombinant NO synthase genes, to investigate the impact of NO production by the bacteria. We show that S. coelicolor ΔnsrR-hmpA is significantly more competent at colonising T. aestivum rhizosphere compared to the control (P<0.005). Endosphere colonisation is sporadic for both mutant and control, this observation is supported by fCLSM imaging. Preliminary data indicates that the Streptomyces strains engineered to express NO synthase at high levels, colonise poorly. This suggests that NO is a dynamic and finely tuned signalling molecule. We are excited to present promising new evidence to support an as yet undescribed link between NO and plant root colonisation by Streptomyces coelicolor. Understanding the mechanisms that underpin this process is the first step in exploiting these interactions for agricultural technology.

The study of animal digestive tracts reveals important information on the host’s health status. For livestock, being able to predict the effect of different treatments on the gut microbiome has important implications for increased sustainability, enhanced animal welfare and increased food safety. However, gut contents can be investigated only after the slaughter of the animal, but cloaca/rectal samples may be collected from live animals and reduce the number of animals killed for experimental purposes. The aim of this study is comparing the microbial communities of caecum and cloaca associated with eight poultry broiler flocks from two English farms. 16S amplicon libraries were run on a MiSeq with a 250 bp PE read metric. The data were evaluated with in qiime1 and qiime2. Comparisons of bacterial communities of cecum and cloaca revealed they are significantly different in terms of the number and types of bacterial species, as well as their abundance (P-value Indicator species analysis of cecum samples showed the class Bacilli were enriched, while Clostridia had greater prevalence in cloaca. Finally, no pathogenic bacterial species of poultry were identified in the analysed animals. Despite the fact sampling cloaca content could be a method to reduce cost and suffering for research purposes, this study reveals the limit of the use of cloacal microbiomes to provide a window into poultry alimentary canal microbiomes.

Anti-virulence strategies are an alternative approach to combat zoonotic bacterial pathogens. Although control measures are in place against E. coli O157, it still remains a global health concern with cattle being the most important reservoir. Different feed constituents have varying effects on the virulence of E. coli O157 present in the gut which then are transferred to meat processing surfaces and meat during slaughter. This study explored the anti-virulence properties of a mixture of natural plant extracts and organic acids (citrus, grape and oregano extracts, lactic and citric acid) before and after refrigerated storage as means of reducing the risk from E. coli O157 by reducing its virulence. Assessment of the effects of sub-inhibitory concentrations (0.1 and 0.5 %, v/v) of the antimicrobial mixture before refrigerated storage showed that the pathogen’s motility and adhesion onto HCT-8 cells was significantly reduced (P<0.05) in a dose-dependent manner. Shiga-toxin 2 production was also significantly reduced. Real-time PCR analysis revealed that the mixture of natural antimicrobials repressed expression of adhesion (eae) and shiga-toxin 2 (stx2) genes, which was consistent with the observed reduction in adhesion and toxin production. The same virulence factors were investigated after simulated storage of E. coli O157 in meat simulation medium. Results revealed that after exposure to the antimicrobial mixture, the virulence was significantly lower compared to non-treated control after refrigerated storage. The present work shows the potential of the antimicrobial mixture in reducing virulence and thus risk from E. coli O157 by applying it as a feed additive.

Excess application of Nitrogen (N) to agricultural soils can lead to environmental pollution. As nitrous oxide (N₂O) is a potent greenhouse gas, it is of critical importance to reduce its emission for climate change mitigation. Availability of nitrogen can facilitate denitrification, an anaerobic respiratory pathway carried out by microbial communities in which N₂O is an intermediate product. An understanding of soil, climatic and edaphic factors influencing microbial communities and their activity is key to reducing N₂O emissions. Soil pH strongly impacts microbial community structure, with a direct effect on NosZ, the pH-sensitive enzyme catalysing N₂O reduction. We thus expect that microbial communities in acidic soils have a reduced capacity to mitigate N₂O emissions. It is likely that other management factors, like phosphorus application, interact with pH; causing changes to chemical nutrient availability and direct effects on microbial composition. The complexity linking N₂O emissions and microbial activity (impacted by soil pH), and the interacting role phosphorus availability plays in this relationship, is not yet understood. In this study, the capacity of microbial communities to denitrify, as well as the functional microbial community impacted by soil pH, were analysed by potential denitrification assays, measuring N₂O fluxes and N₂O/(N₂O+N₂) ratios; and qPCR analysis of denitrification genes. This was investigated across two soil types with a pH gradient and range of phosphorus application rates. Understanding the link between the microbial communities and N₂O production can be applied in agricultural management to reduce emissions from fields.

Viable but non-culturable (VBNC) cells are cells that are metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can ‘resuscitate’ restoring their ability to grow on media. Currently, over 80 bacterial species are reported to enter the VBNC state. The ability of VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in pathogens may pose a risk to human health and thus warrants further investigation. This research project has investigated the ability of the human pathogen Vibrio parahaemolyticus to form VBNC cells when exposed to stressful conditions. V. parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide and often results in watery/bloody diarrhea and vomiting. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and report that different sub populations of VBNC cells can occur based upon their metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella. Using mass spectrophotometry we have identified several proteins which may play roles in VBNC formation and resuscitation in V. parahaemolyticus.

Screening of an Antarctic soil functional fosmid metagenomic library identified a novel bacterial gene, homologous to known Water Hypersensitivity (WHy) domains. The WHy domain is a typical component of Late Embryogenesis Abundant (LEA) proteins which occurs widely in both prokaryotes and in plant eukaryotes and are expressed under various stress conditions [1]. A phylogenetic analysis of multiple WHy homologues from different species suggested that the ancestral origin of this protein gene lies within the ancient archaea [1]. Our previous studies have shown that this bacterial protein elicits significant protection against freeze and cold stress in recombinant E. coli [2]. Expression of the WHy gene in Arabidopsis resulted in a wide range of statistically significant stress-tolerant phenotypic properties. These included an increase of up to 6-fold higher germination efficiency of transgenic recombinant seeds compared to the WT, and a 100 % survival rate of WHy gene-expressing plants compared to 0 % survival of adult WT plants after freeze shock. Similar improvements in survival rates were observed for recombinant plants in drought stress experiments.

Commercial poultry rearing systems often house successive flocks of birds with limited between-flock cleaning of the poultry houses. Previous research focused on opportunities for successive flocks to become colonised with pathogenic bacterial species. However, there is a paucity of information regarding the transfer and persistence of commensal bacterial between flocks, and if this might confer health benefits on subsequent flocks. The work presented here utilised 16S community sequencing to characterise the development of the microbial flora of commercially reared broiler chickens and turkeys to determine microbial environmental persistence. DNA was isolated from caecal contents, faeces, and various bedding samples collected from nine sites over a nine month period. Samples were taken from houses used for rearing chickens or turkeys or where alternating chicken and turkey flocks were reared. Measures of alpha diversity for the different samples suggested that both chickens and turkeys had a similar microbiota. Unsurprisingly, members of the microflora could also be found in the environmental samples tested, although survival was dependent on the phyla and bedding material. Further analysis of the samples is currently underway, in order to determine the extent, if any, of microbial transfer between flocks, with a particular focus on potential poultry microbiota species differences. This study demonstrated how commensal microbes are able to persist within poultry rearing sheds and if this transfer impacts on subsequent flock performance. Alongside increasing understanding of microbial environmental persistence, the work also shows how effective current biosecurity methods are in controlling the transfer of all microbes, including pathogens.

The overall value of Irelands beef exports is worth approximately €2.5bn which is an annual increase of 5 % in 2017. However, the beef industry faces many challenges to export products to distant markets including a short shelf life and other economic losses that are mainly caused by microbial contamination. One of the simplest approaches to limit this contamination on the surfaces of beef carcasses is to use alternative carcass chill regimes. Carcasses underwent an industry standard chill process (10 °C for 10 h followed by 0 °C for 38 h) and were compared with carcasses that underwent a more rapidly chilled process (0 °C for 5 h and −2 °C for 3 h). Bacterial concentrations (mesophilic and psychrophilic total viable counts, total Enterobacteriaceae counts, Lactic Acid Bacteria, Pseudomonas spp., Brochothrix thermosphacta, Clostridium spp.), physiochemical (pH, temperature, water activity (aw)) and organoleptic (colour, odour, texture) changes were monitored throughout the entire beef food chain (carcass → primal → retail steak) until end of shelf life. Rapidly chilled carcasses had significantly (P<0.05) less bacterial surface contamination compared to conventionally chilled carcasses. There was also significantly (P<0.05) less evaporative loss on carcasses and primals which will have a positive economic impact on the beef industry. This longitudinal study is one of the largest trials ever performed on beef shelf life extension.

Phoma stem canker is one of the most important diseases of winter oil seed rape (Brassica napus) world-wide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and Leptosphaeria biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double stranded (ds) RNA virus in L. biglobosa and no viruses in L. maculans. The virus forms isometric particles ca. 40–45 nm in diameter and has four genomic segments, each possessing a single open reading frame flanked by untranslated regions. Phylogenetic analysis revealed modest similarities to known and suspected members of the family Quadriviridaeand therefore the virus was nominated Leptosphaeria biglobosa quadrivirus-1. Following eradication of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance towards L. maculans in B. napus following lower leaf pre-inoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defences. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of phoma stem canker later in the growing season.

Recent landmark studies indicate that the nucleolus plays key roles in stress responses including the DNA-damage response (DDR). The latter involves interactions of components of the DDR machinery including NBS1 with the sub-nucleolar protein Treacle, a key mediator of ribosomal RNA (rRNA) transcription and processing, implicated in Treacher-Collins syndrome. Using comparative proteomics, confocal and single molecule super-resolution imaging, and infection under BSL-4 containment, we have shown for the first time that the nucleolar DDR pathway is targeted by infectious pathogens [1]. We found that the matrix (M) proteins of Hendra virus and Nipah virus, highly lethal viruses of the Henipavirus genus (order Mononegavirales), target Treacle to inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhanced henipavirus production. These data identify a novel mechanism for viral subversion of host cell biology by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intra-nucleolar function for proteins of any mononegavirus [1, 2]. For the presentation I will discuss our new data, which is advancing our understanding both of the mechanisms impacted by the Henipavirus-Treacle interaction, and potential roles of such interactions in infection by other viruses, including highly lethal lyssaviruses [3].

[1] Rawlinson et al. Nature Communications 2018.9 : 3057 (2018)

[2] Rawlinson et al. Cellular Microbiology 2015. 17(8):1108–20

[3] Oksayan et al. Journal of Virology 2015.89(3):1939–43