- Volume 1, Issue 1A, 2019

Helicobacter pylori is the leading cause of peptic ulcers and gastric cancer. Eradicating H. pylori infections is becoming more difficult due to increasing antibiotic resistance and poor patient compliance. We aim to develop a novel liposomal drug delivery system that encapsulates antibiotics and the antimicrobial fatty acid linolenic acid (LLA). We hypothesise that H. pylori will have much lower resistance rates to this dual formulation. Liposomes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin, and cholesterol were produced using the thin film hydration method. The size of our liposomes was assessed using dynamic light scattering (DLS), and their antimicrobial activity was assessed using a viable count assay. The liposomes had a particle size ranging from 95 to 150 nm. We have successfully loaded LLA up to 20 % of total lipid composition, and shown stability of the particles in storage at 4 °C for up to 3 months. The LLA liposomes achieved complete eradication of H. pylori in vitro at 150 µg ml−1, whereas control liposomes containing no LLA had no antimicrobial effect. We have recently formulated liposomes encapsulating LLA and amoxicillin at 2.2 mg ml−1 and 0.99 mg ml−1 respectively, and are currently assessing their antimicrobial activity against H. pylori. In conclusion, it is possible to prepare LLA- and amoxicillin-containing liposomes and LLA has antimicrobial activity against H. pylori. In future work we will utilise strategies to enhance gastric retention which will provide local bactericidal activity, improving on inefficient systemic uptake mechanisms with current therapies.

Clostridium difficile is the primary cause of antibiotic associated diarrhoea globally. In the UK there has been a decline in the prevalence of C. difficile due to implementation of surveillance and infection control procedures. At Rideout Hospital, USA, however, there is a high incidence of C. difficile infection, which has been partly attributed to poor infection control measures. Other factors include the ability of spores to adhere to fomites such as surgical gowns. It has been demonstrated that the single-use polypropylene surgical gowns used at Rideout can ‘trap’ hydrophobic epidemic spores of C. difficile within the fibres, which can then be transferred to stainless steel surfaces and hospital floor vinyl; even with use of appropriate sporicides such as sodium dichloroisocyanurate. This study sought to establish the strains of C. difficile present on the gowns and thus inside the nosocomial environment. Contaminated gowns from Rideout were cultured for 5 days anaerobically in Brain –Heart Infusion broth supplemented with 0.1 % Sodium taurocholate. Broth culture was screened for the presence of C. difficile using CCFA media, C. DIFF QUIK CHEK COMPLETE®, 16 s-23s RNA analysis and toxin PCR. Once isolated, strains were sequenced and tested for biocide susceptibility to in-use concentrations of Sodium dichloroisocyanurate. In total 23 suspected C. difficile samples were isolated from the gowns; of which 8 were confirmed. Sporicide susceptibility testing is ongoing. Once infective strains have been identified measures can be taken to enforce appropriate infection control procedures in order to limit the prevalence of spores and reduce infection rates.

Chronic wounds (CW) are a common complication of diabetic ulcers (DUs), which are a major burden to health care systems worldwide and can result in lower limb amputation due to the intractability of the infection. In DUs there is a high probability of the infecting bacteria evolving considerable phenotypic and genetic diversity, as has previously been shown for chronic lung infections. However, it is not known whether this is also the case for chronic DUs, and whether diversity impacts on virulence and antibiotic resistance. To study this, bacterial populations were isolated from different samples from patients with DUs. Phenotypic diversity was investigated in P. aeruginosa populations through the analysis of phenotypes traditionally associated with pathogenicity, and through a whole genome study. Phenotypic variation in P. aeruginosa isolates taken from different patients was observed, but little variation within the same CW (with exception of one patient with a leg ulcer). Antibiotic resistance was found to increase during the course of infection, and it became apparent that P. aeruginosa colonisation in DUs is via a single strain per ulcer, and potentially per patient, even though some sample-specific phenotypic profiles were found to arise from a homogenous population. For this case, a detailed genomic analysis between bone and blood isolates was done, including a comparison of their transcriptomes using RNAseq. The results suggest that the loss of flagellum facilitated evasion of the innate immune system, which allowed bacteria to go undetected and spread systemically causing the rapid decline in the patient’s health.

Antibiotic is a formidable remedy to infections caused by diverse microbial agents. This assertion is however questioned in the wake of antimicrobial resistance. Fifty clinical isolates of Pseudomonas aeruginosa were obtained from both in and out-patients using standard procedure. The isolates were identified using standard biochemical tests. The antibiotic susceptibility pattern of each isolate was examined inaccordance to the Clinical and Laboratory Standards Institute (CLSI) guidelines using the Kirby-Bauer’s disc diffusion method. The antibiotics used in the study includes: Ciprotab, Colistin-sulphate, Meropenem, Ceftraxone and Cefepine. Out of the clinical isolates obtained, a total of 48 per cent male and 52 per cent females were the population under study. The percentage ratio of in-patient and out-patient examined were 32% to 68 %. The percentage distribution of the administration class for medical and surgical was 34% and 66% respectively. The highest incidence of Pseudomonas aeruginosa was from patients that have undergone cesarean section (28%). Highest susceptibility was observed in Ciprotab (82%) Meropenem (64%) and Ceftraxone (46%). Highest number of resistance was observed against Cefepine and Colistin Sulphate while less than 5 % were resistant to Ciprotab and Meropenem. Meropenem and ciprotab were the two classes of drugs that showed highest activity against Pseudomonas aeruginosa. Commonly used antibiotics must be continuously examined for its efficacy. Anti-microbial susceptibility monitoring is necessary inorder to guide physicians in prescribing the right combinations of anti-microbials to limit and prevent the emergence of multi-drug resistant strains of P. aeruginosa.

Pediatric diarrheal disease presents a major public health burden in low- to middle-income countries. The clinical benefits of empirical antimicrobial treatment for diarrhea are unclear in settings that lack reliable diagnostics and have high antimicrobial resistance (AMR). In this study, conducted a prospective multicenter cross-sectional study of pediatric patients hospitalized with diarrhea containingblood and/or mucus in Ho Chi Minh City, Vietnam. Clinical parameters, including disease outcome and treatment, were measured. Shigella, nontyphoidal Salmonella (NTS) and Campylobacter were isolated from fecal samples, and antimicrobial susceptibility profiles were determined. Statistical analyses, comprising log-rank tests and accelerated failure time models, were performedto assess the effect of antimicrobials on disease outcome. Among 3166 recruited participants (median age 10 months; interquartile range, 6.5–16.7 months), one-third (1096 of 3166) had bloody diarrhea, and 25 % (793 of 3166) were culture positive for Shigella, NTS, or Campylobacter. More than 85 % of patients (2697 of 3166) were treated with antimicrobials; fluoroquinolones were the most commonly administered antimicrobials. AMR was highly prevalent among the isolated bacteria, including resistance against fluoroquinolones and third-generation cephalosporins. Antimicrobial treatment and multidrug resistance status of the infecting pathogens were found to have no significant effect on outcome. Antimicrobial treatment was significantly associated with an increase in the duration of hospitalization with particular groups of diarrheal diseases. Our results imply a lack of clinical benefit for treating diarrhea with antimicrobials in a setting using high antimicrobials; adequately powered randomized controlled trials are required to assess the role of antimicrobials for diarrhea.

An accurate and rapid identification of Candida species in cancer patients with pulmonary symptoms can provide important information for effective treatment. Candida infections represent an increasing cause of morbidity and mortality in patients receiving immunosuppressive chemotherapy for cancer, organ transplantation or in immunocompromised. We used conventional methods such as culture, fermentation reactions, morphology and molecular methods based on the ribosomal DNA repetitive regions or the Internal Transcribed Spacer (ITS) for the identification of Candida species. Seventy bronchial specimens of Bronchoalveolar Lavage (BAL) from cancer patients at Shaukat Khanum Memorial Cancer Hospital and Research Center. Lahore, Pakistan were included in this study. Seventy cancer patients were diagnosed on the basis of histological profile. Candida detected by conventional methods using Sabouraud Dextrose Agar (SDA), potassium hydroxide (KOH) preparation, germ tube test, fermentation reactions and Gomori methanamine-silver stain (GMS). Thirty (42 %) positive isolates of Candida species were obtained by culture, twenty (28 %) isolates were germ tube test positive while thirty isolates (42 %) were positive by PCR method. In conclusion, the results of our study showed that the PCR based detection methods are significantly better and can detect Candida with more accuracy and specificity as compared to conventional methods. Our study would pave the path for optimization of protocols for detection of Candida in cancer patients.

Candidiasis is one of the most prevalent mycoses worldwide, and there are few successful treatments for this disease. In the UK, Candida glabrataaccounts for 25 % of Candida infections, and due to the increasing incidence of multidrug resistance, C. glabrata poses an eminent threat to public health, exacerbated by the limited range of antifungal therapies. Our aim is to elucidate the molecular mechanisms underlying the emergence of pathogenicity in the C. glabrata lineage, to identify novel therapeutical targets. Using comparative phylogenomics, we putatively identified 19 genes under positive selection in the C. glabrata lineage. Each of these genes influences chromatin structure by regulating histone post-translational modifications (PTMs). To assess the contribution of these genes to virulence, we monitored the phenotypic consequences of individually removing each gene in clinically-relevant assays. To date, we have focused on 3 genes, cg-SPP1 (regulates histone H3 methylation), cg-HAT1 (histone H4 acetyltransferase) and cg-AHC1 (subunit of Ada histone acetyltransferase complex). Preliminary data show that the individual deletion of these genes increases biofilm formation and fluconazole resistance in C. glabrata. Furthermore, acetyltransferase knockout strains show hypervirulence in an in vivo Galleria melonella infection model, and phenotypic differences in abiotic stress assays. RNA-sequencing was performed on the type-strain, cg-hat1Δ, and cg-ahc1Δ, in the presence and absence of fluconazole to determine the molecular bases for these phenotypes, revealing commonality between differentially expressed genes in both mutants. Together, our data suggest that histone PTMs play a significant, and overlapping, role in dictating virulence in C. glabrata, and detail the global transcriptomic response to fluconazole.

Dinoflagellates are important marine algae, being an essential symbiont in corals. Loss of the dinoflagellates causes coral bleaching, and ultimately death of coral reefs. Current efforts to study dinoflagellates are greatly hindered by the lack of a reliable method of genetic transformation. The chloroplast genome of dinoflagellates is fragmented into multiple plasmid-like minicircles, each carrying at most a few genes. We have used ‘artificial’ minicircles, based on fusions between endogenous minicircles and E. coli plasmids to establish a transformation system and optimize parameters for it. We have determined the sensitivity of wild type dinoflagellate strains to possible selective agents. We have compared the performance of a number of transformation methods, including electroporation and particle bombardment, and find that electroporation is not effective for transformation, whereas particle bombardment (‘biolistics’) is. We have assessed different parameters for biolistics and subsequent selection. We have shown the successful maintenance of sequence from an artificial minicircle within a dividing cell population over a period of six months, as well as evidence (using RT-PCR) of transcription and the expected phenotype for the inserted gene. This represents a significant step forward in developing the genetic modification of Amphidinium carterae. We are currently testing the protocol with other dinoflagellates.

Dinoflagellates nuclei are unlike any other. They have: 1) highly inflated genome sizes, 2) practically lack histones and nucleosomal organization; 3) have permanently condensed liquid crystalline chromosomes throughout the life cycle, and; 4) contain a novel a major new nuclear DNA-binding protein. This protein, called Dinoflagellate/Viral NucleoProtein (DVNP) is small in size (10–20 kDa), highly positively charged (30–40 % R+K), and its gene is one of the most highly transcribed in dinoflagellate cells. It has no homology to histone proteins, has no homologues in either eukaryotes or prokaryotes, but is found in a number of marine large DNA viruses. To understand the role of DVNP in the dinoflagellate nuclei we have expressed and purified DVNP and are studying the properties of this novel protein and its interaction with DNA. We show that DVNP is a monomer in solution, but upon exposure to DNA it rapidly binds to and compacts DNA into complexes micrometers in size. Using single-molecule imaging and optical tweezers, DVNP is seen to compact DNA a rates of over 50 µm/sec and change the mechanical properties of DNA. Most interestingly, the DVNP/DNA aggregates show a propensity to travel along the DNA strand en masse. Together, these observations suggest that DVNP plays a central role in the novel model for chromatin management found in dinoflagellates.

Narnaviruses are a group of single-stranded, positive-sense RNA viruses, which are non-encapsidated and hence normally transmitted vertically. Narnaviral genomes encode a single protein: the RNA-dependent RNA polymerase (RdRp), which catalyses viral replication. Currently, there are just two recognised species within the genus Narnavirus, both of which are known to infect Saccharomyces yeast. Here, we systematically identify and characterise narnaviral genomes in public sequence databases, using a combination of in silicoapproaches. We identify two major clades of narnaviruses, and propose the establishment of a taxonomic framework based upon their molecular characteristics. Codon usage bias across both clades was analysed and compared with those of potential host taxa from across the eukaryotic domain of life. In one clade, we demonstrate the widespread presence of a long reverse-strand open reading frame (rORF), which typically occupies >90 % of the full-length genomic RNA. Comparative analysis shows that the putative rORF-encoded proteins are highly divergent in amino acid composition, with a central region of increased conservation. These findings shed new light on one of the most divergent clades of eukaryote-infecting viruses.

Diabetic foot ulcers (DFUs) are slow healing wounds which arise from co-morbidities associated with diabetes. Often these ulcers become infected leading to gangrene, osteomyelitis and sepsis. Current treatment options include debridement and a topical irrigant which have limited success. Understanding the effects that these treatments have on microbiome of DFUs and on wound healing is poorly understood. This study compared the efficacy of two irrigant solutions (Prontosan and Electrolysed water –E.W.) on their impact on the DFU microbiome, their role in DFU healing and their effect on biofilm viability. Sequential samples taken from 7 patients undergoing treatment with either one of the irrigants, over a 4 week period revealed commonly observed genera present included Staphylococci (96 %), Propionibacterium (96 %) and Finegoldia (89 %). A unique composition and diversity was observed in the microbiome of each individual DFU. Increasing microbial diversity within the DFUs was correlated with an elevated percentage abundance of anaerobic and Gram negative genera whilst inversely correlated with facultative anaerobic and Gram positive genera. No significant reduction in diversity or species richness of the DFU microbiomes was observed after treatment with either irrigant. Both Prontosan and E.W. had similar effects upon S. aureus biofilms reducing viability by 82.013 % and 86.89 % respectively however E.W. efficacy was strain specific. In addition, E.W. was ineffective at preventing biofilm formation in 6/8 (75 %) S. aureus strains. Better understanding of the DFU microbiome and investigations into novel therapies is paramount to aid our ability to improve the quality of life for diabetic patients.

Although fungi are fundamental to the human microbiome, the diversity and dynamics of the mycobiome is poorly understood, particularly in considering their association with infectious disease, autoimmune disorders and atopy that affect immunocompromised individuals and infants. Characterising the human mycobiome faces several challenges relating to their low abundance and lack of standardized procedures for sample collection and isolation of viable cells and/or quality genetic material for culture-dependent and independent taxonomic and functional characterisation. To address these issues, we have developed a mycobiome analysis pipeline employing both culture-dependent and independent methods to identify as well as isolate, where possible, the fungal taxa populating the human intestinal tract. In a proof-of-concept study this pipeline has been used to identify fungal populations in faecal samples obtained from a small cohort of young infants, aged 2 years or younger. All were born prematurely, and severely immunocompromised and at risk from invasive and potentially lethal microbial infections, including those caused by fungal overgrowth. We have used this combined approach successfully to identify the fungi present in each individual infant, and to recover viable isolates. To date, Candida albicans and C. parapsilosis are the most frequently isolated fungi. While both are major opportunistic human fungal pathogens, C. parapsilosis is particularly problematic to preterm babies, due to its innate ability to form biofilms. Detailed characterisation of these isolates is currently underway. Two large-scale longitudinal microbiome studies have started at the Quadram Institute, and our validated analysis pipeline will be incorporated to define the fungal component of each study participant.

The surface associated communities of microorganism in biofilms are encased in a matrix of extracellular polymeric substances (EPS). The EPS is made up of mainly polysaccharides, proteins, nucleic acids and lipids and plays an important role in maintaining the integrity of the biofilm (1). Although the general composition of the EPS is known, it can be highly variable among strains and among different growth conditions for the same strain. Due to the large variety of biopolymers in nature and the difficulty in their analysis, EPS has been called ‘the dark matter of biofilms’ (2). In order to develop a comprehensive understanding of the matrix of the biofilm, EPS was extracted from four Pseudomonas spp., mCherry-expressing Pseudomonas fluorescens, GFP-expressing Pseudomonas putida and the wild types of Pseudomonas fluorescens and Pseudomonas putida. The extractions were carried out on biofilms grown on glass slides using the cation exchange resin (CER) method. Colorimetric methods were used to quantify the sugars and proteins present in the EPS. These colorimetric assays showed that there was a larger amount of proteins present compared to sugars. The proteins present in all four biofilms of Pseudomonas spp. were identified by LC-MS/MS while NMR and HPLC were used to identify the sugars present. The knowledge gained by these results have the potential to aid in the development of biofilm eradication methods through the targeting of specific components of the EPS. 1. Starkey, M., et al. (2004), American Society of Microbiology: 174–191.2. Flemming, H.-C. and J. Wingender (2010). Nature Reviews Microbiology 8 : 623.

The use of engineered nanoparticles (NPs) as a technique for antimicrobial delivery aimed at biofilm treatment is an emerging field of research. There have been numerous studies involving a wide range of NPs showing varying results regarding anti-bacterial effects (1). However, research focusing on specific interactions between functionalized nanoparticles and the extracellular polymeric substances (EPS) of biofilms are limited. The complexity of the biofilm matrix may be hindering the understanding of the fundamentals which govern biofilm – nanoparticle interactions. There are a wide range of physicochemical properties which influence the uptake and retention of nanoparticles within the matrix including NP size and charge properties, biofilm topography and porosity and EPS composition (2). These aspects must be considered when studying biofilm – nanoparticle interactions. In order to identify these specific interactions, a series of experiments were carried out using mCherry-expressing Pseudomonas fluorescens and GFP-expressing Pseudomonas putida biofilms. Using high throughput fluorescent intensity measurements and confocal microscopy, it was possible to investigate the uptake of surface functionalized silica NPs by the two biofilms and obtain valuable information regarding biofilm – nanoparticle interactions. The results suggest that specific NP surface functionalization has a major role in guiding the interaction and binding of EPS components, possibly due to electrostatic interactions between NPs and the EPS. The findings of this research will help with the future design of nanoparticles with specific modes of action towards components in the EPS.1. Ramos M., et al. (2018). International Journal of Nanomedicine13 : 1179–1213.2. Nevius BA., et al.(2012). Ecotoxicology21 : 2205–2213.

The ocean is home to a huge diversity of life that perform a wide range of functions key to earth systems. Marine algae communities contribute a significant proportion of net carbon fixation of the globe as well as forming the basis of the food webs for every trophic level above their own. Despite their importance, many of the taxa that make up these microalgal communities are relatively unknown and understudied, due to their recalcitrance to lab culturing. This concept is known as microbial dark matter and applies in particular to many non-photosynthetic lineages which have been long overlooked by the research community. To investigate whether dependencies of these taxa on certain B vitamins may play a role in this unculturability, data sets produced form the Tara oceans expedition will be analysed. The Stramenopile lineage will be analysed in greater detail for their metabolic potential to synthesise the B vitamins, and/or whether they may dependent on an external source. Both Single-Amplified Genomes (SAG) and metatrascriptomic data sets will be analysed to determine the complement of genes present in different Stramenopile lineages. This will then be correlated with the global distribution data attainable from analysing Tara data sets. By this means, we hope to understand more about the metabolic contribution of this taxonomic group to the community, how this supports the community as a whole and whether dependence on B vitamins is a contributing factor to unculturability.

Difficulties in the removal of bacterial biofilms in the industrial and biomedical sectors have driven the development of new technologies. Although numerous studies have highlighted the use of nanoparticles (NPs) as antibiofilm agents, the fundamental physicochemical interactions between NPs and the biofilm matrix is still poorly understood 1. The development of ‘smart nanoparticles’ for biofilm removal requires an in-depth understanding of the complex interactions between NPs and biomolecules within the extracellular polymeric substances (EPS) of the biofilm matrix. These interactions are highly dependent on the physical and chemical properties of the NPs 2. In order to identify and characterize the specificity of binding and the direct interaction between silica NPs (SiNPs) and EPS matrix components of Pseudomonas spp. biofilms, a range of experiments were carried out. Biofilms were exposed to SiNPs of different sizes, charges and surface functionalization while biomolecules such as proteins, polysaccharides, and eDNA were fluorescently labelled and their distribution, relative abundance and their colocalization with SiNPs within the biofilm was quantitatively assessed using CLSM microscopy. Changes to the SiNPs size and surface-chemistry dramatically affected their interactions with biomolecules in the biofilm matrix. This includes the increased affinity (or interaction) of SiNPs to preferentially bind to proteins and beta-linked polysaccharides and also lead to changes in the degree to which aggregation of SiNPs occurs within and on the surface of the biofilm.

1. Ikuma K et al. (2015). Front. Microbiol. 6, 591

2. Bewersdorff, et al. (2017). Int. J. Nanomed. 12, 2001–2019

100 years have passed since the independent discovery of the humble bacteriophage (phage) by Frederick Twort and Felix d’Herelle in 1915 and 1917 respectively, and since then, it has become commonly accepted that phages represent the most abundant biological entities on Earth. Despite this fact, viral taxonomy lies in extremely treacherous waters, ever changing to accommodate the next series of phylogenetic mysteries. The utilisation of genes such as the terminase large sub-unit can in some cases provide a robust taxonomic marker, but this is often found to fail at higher taxonomic levels. In addition, the rapid evolutionary dynamics and highly modular nature of phages provide yet more phylogenetic roadblocks, necessitating additional and multifaceted approaches as a means of resolution. Here, we describe a novel approach towards the taxonomic classification of phage systems. Tools for accurately predicting the three dimensional structure of proteins are improving at an unprecedented rate due to the fact that the number of protein sequences far exceeds the number of experimentally determined structures. Our approach leverages these methods through a pipeline which compares models of phage marker genes in order to permit the inference of phylogenetic relationships based on cross model superimposition. We hope this method will supplement other approaches in providing a more holistic approach to viral classification.

The imminent threat of antimicrobial resistance has necessitated that the search for novel antimicrobials be widened to lesser-explored environments. Marine and freshwater sponges have emerged as the most prolific source of such compounds over the last decade, representing the most widely sampled phyla in the hunt for novel biologics over the last 45 years. Most of the work however has focused on sponges from shallow waters, with the deep-sea sponge microbiome highlighted as a major source of untapped antimicrobial potential. Optimisation of bacterial recovery was carried out for two previously unstudied species of deep-sea Hexactinellid sponge species (Pheronema carpenteri and Rhabdodictyum delicatum recovered from the Rockall Trough, North Atlantic), using a variety of culture media, supplementation and environmental conditions. This optimisation was carried out in parallel with 16S rDNA metagenomic sequencing in order to determine community composition for both sponge species (IonTorrent, Life Technologies). All recovered isolates were assayed for antimicrobial activity, forming a panel of ‘active’ organisms. Two isolates (Ph16-28; A11) were selected for downstream purification and characterisation of the responsible antimicrobial agent via column chromatography. Isolate identities are currently being confirmed via draft whole-genome sequencing (MinION, Oxford Nanopore), and are suspected to be members of the Bacillus and Streptomyces genera. Current data provides a working axiom for the cultivation of deep-sea sponge microbes and suggests the deep-sea sponge microbiome to be a promising source for novel antimicrobials.

Contemporary metagenomic annotation methods have proven insufficient in our attempts to better understand the complex environments around us. We call the yet to be annotated part of a metagenome it’s ‘dark matter’. The Gene Ontology (GO) is a hierarchical vocabulary used to describe gene product function and a large collection of curated genes with GO annotations already exists. DeepGO utilises deep learning to build models from these curated genes and gene products to predict GO categories for novel proteins. One of the major problems with metagenomic studies today is the process of assembling the environmental DNA sequences into their original genomes. This is difficult, with chimeric metagenomically assembled genomes being common. To avoid this and the computational and time expense, we have modified DeepGO to perform protein function prediction directly from sequence reads with limited protein coding sequence prediction. Three independent models were trained as the following; The first 50 amino acids of a protein were used for training, The last 50 amino acids were used for training, A phasing window of 50 amino acids was used to train across the entirety of a protein sequence. These models were chosen to learn from the different parts of a protein sequence we are likely to capture from only the short unassembled sequence reads. We compared the three models by producing a mock metagenomic community consisting of 6 model bacterial genomes. We evaluated the functions predicted from the unassembled sequence reads and the protein coding sequences predicted from the assembled metagenome.

The rumen bacteria play a major role in lipid metabolism. Bacteria remove the double bonds of unsaturated fatty acids resulting in the production of saturated fatty acids, which are incorporated in milk. Crude olive oil (rich in unsaturated fatty acids) represents a potentially valuable feed source for dairy cows that might enhance the human-health beneficial composition of milk and dairy products. This project studied the effect of supplementing dairy cow diets with olive oil (OO) and palm oil (HVO) on rumen microbiota. For 63 days the animals were fed a control diet (basal diet) with no added lipid and two fat-supplemented diets (30 g kg−1 DM). Rumen sampling were performed at the onset of the experiment and every 21 days for 63 days using a rumen scoop. Total microbial DNA was extracted from ruminal samples for high-throughput sequencing of the 16S rRNA gene through Illumina MiSeq platform. Results revealed the dominance of phyla Firmicutes and Bacteroidetes. Firmicutes was the most prevalent phyla in diet control (75.2 %), OO (71.1 %) and HVO (75.2 %). At genus level Succiniclasticum and Prevotella were the dominant genera, belonging to Firmicutes and Bacteroidetes respectively. Succiniclasticum decrease significantly their relative abundance during OO supplementation (p)

Improved amylases were developed from protoplast fusants of two amylase-producing Apergillusspecies. Twenty regenerated fusants were screened for amylase production on Remazol Brilliant Blue agar. Crude enzymes were produced by solid state fermentation on rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized for amylase activities of parent and selected fusants on a rice bran medium by solid state fermentation. The variables assessed were optimized using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activities at room temperature and at 80 °C showed Aspergillusdesignates, T5 (920.21 U ml−1, 966.67 U ml−1), T13 (430 U ml−1, 1011.11 U ml−1) and T14 (500.63 U ml−1, 1012.00 U ml−1) as preferred fusants. Amylases produced by the fusants were observed to be active over the range of pH studied. Fusants T5 and T14 had an optimum acidic and alkaine pH respectively. Optimization studies revealed enzyme T5 at pH 4 and temperature of 40 °C as optimum for amylase production. The statistical tools employed, predicted and compared the optimal conditions for enzyme activities of amylases from parent and fusant strains of Aspergillus revealing the desirability of the fusants over the parents in industrial applications.

Aflatoxin M1 is a metabolite of the most potent aflatoxin, (AFB1) and thus, has been treated and rendered not so toxic, on this basis, its study has been taken for granted. It is the aim of this study to ascertain the toxic nature of AFM1 by determining its effects on microbial flora in the gut of neonatal rats. A dosing experiment was conducted on the neonates, where they were divided into groups and treated with different concentrations of AFM1 using uncontaminated milk as a carrier medium into the rats. The rats were sacrificed; the small and large intestine were harvested and cultured on appropriate selective media for growth of microorganisms. Results show samples from the control group had an uninterrupted microbial community, while the treated group, with increasing doses of AFM1 decreases and depletes the microflora in the gut samples. Lactic acid bacteria were also significantly depleted by AFM1. These findings suggest the capability AFM1 in modifying the gut microbiota in a dose-dependent manner which might result in serious health hazards in neonates.

Acetyl-phosphate (AcP), an intermediate from the phosphotransacetylase-acetate kinase (PTA-AK) pathway, has shown to be critical in pathogenic bacteria for the general metabolism and synthesis of virulence factors. Lysine acetylation is a post-translational modification (PTM) that occurs enzymatically and non-enzymatically by the addition of an acetyl residue from acetyl coenzyme A and AcP, respectively. Neisseria gonorrhoeae, the etiologic agent of gonorrhoea, has been shown to use AcP for lysine acetylation, however, the role that AcP has in the pathogenesis and how acetylation is regulated has not been discerned. The concentration of AcP was altered in N. gonorrhoeae MS11 by interrupting the genes involved in the PTA-AK pathway, pta and ackA, and the gene that encodes for a lysine deacetylase family protein, kdac. AcP concentrations were increased in ΔackA and decreased in Δpta resulting in modulation of lysine acetylation. Growth on glucose, lactate or pyruvate were investigated. In aerobic conditions, ΔackA mutant solely grew in glucose, while the Δpta mutant grew in glucose and lactate. In microaerophilic conditions, ΔackA and Δpta mutants solely grew in presence of glucose. The virulence of ΔackA and Δpta was tested by infecting larvae of Galleria mellonella. WT killed 50 % population (n=15) after 6 days and ΔackA after 24 h, however, Δpta after 6 days it only killed 10 %. Taken together, our results show AcP as an important metabolite for the metabolism and virulence of N. gonorrhoeae.

Bacterial strains were obtained from wheat rhizosphere and screened for zinc solubilization on agar plates. Two strains FA-9 and FA-11 were found efficient for Zn solubilizing activity and were identified as Pseudomonas aeruginosa and Enterobacter sp. by 16S rRNA and gyrase (gyrB) genes analysis, respectively. The strain FA-9 produced a clear zone diameter of 63 mm, 60 mm and 51 mm on agar plate amended with different zinc ores. The strain FA-11 produced a zone diameter of 17 mm with zinc carbonate and 20 mm with zinc oxide while no zone was observed with zinc phosphate. Both strains showed no visible activity with ZnS ore. Similarly, FA-9 and FA-11 increased maximum soluble zinc content (102 µg ml−1 and 45 µg ml−1) from zinc carbonate ore as compared to zinc oxide ore (102 µg ml−1 and 45 µg ml−1) in liquid broth. It was noted that both strains exhibited less potential (7 µg ml−1 and 0.57 µg ml−1) to solubilize ZnS ore in liquid broth. A comparison between agar plate assay and liquid broth quantification shows that agar plate assay does not present the solubilizing potential of ZSB precisely. The strains FA-9 and FA-11 produced auxin with l-tryptophan (3.25 µg ml−1 and 2.86 µg ml−1) and without l-tryptophan (1.23 µg ml−1 and 1.02 µg ml−1). Both strains expressed exo-polysaccharides (EPS) and siderophores activity along with phosphate (P) solubilization, ACC deaminase and antifungal activities. The ACC deaminase and N-fixation activity was confirmed by the amplification of acdS and nifH genes respectively.

Urinary tract infection (UTI) is considered to be one of the most prevalent bacterial infections in the world. It affects urinary tract system including bladder and kidney. Uropathogenic Escherichia coli is more prevalent in females due to their anatomical structure as well as they are more susceptible to recurrent infections. Every woman out of three is affected by UTIs. Gram-negative bacteria are a major cause, particularly Escherichia coli (E. coli). E. coli was considered a main causative agent for 80–90 % of community-acquired infection and for about 40 % of nosocomial UTI. Moreover, it is responsible for 25 % of recurrent infection. Proteomic can be used to analyze and identify complete components of proteins. It can be used to distinguish between bacteria based on synthesized proteins. In addition, proteomic is applicable to identify possible targets of therapy. My study aims To compare protein profiles of E. coli from different UTI patients and identify possible unique protein signature for future biomarker studies. Six Urine samples with E. coli were taken from females aged from 15 to 50 years old. Samples loaded onto Sodium dodecyl sulfate Polyacrylamide gel electrophoresis for separation. Samples with abundant proteins profiles on SDS-PAGE, were selected for run on two dimensional gel electrophoresis. Gels were compared to each other to look for interesting protein spots. Many differences were observed in protein profiles of E. coli isolates in both 1D SDS-PAGE and 2DGE. Two bacterial proteins identified as possible candidate were (OmpA) found in Gram negative bacteria and RNA polymerase-binding transcription factor DksA mostly found in E. coli

Vibrio cholerae experiences frequent feast and famine conditions. In the absence of all the three nutrient components (Carbon, Nitrogen, and Phosphorous), the cells could manage starvation and survive up to 6 months with only 1 log reduction in viability. Addition of carbon or nitrogen to the starvation media resulted in 3 log reduction in cell number. Simultaneous addition of carbon and nitrogen (Phosphorous starvation) reduced the cell viability to a below detectable level. The suboptimal growth conditions; non-metabolizable source of carbon and nitrogen; variable C: N ratio; inhibition of metabolism and cell division, increased the cell survival under phosphate starvation. However, when all the three components are limited, the cells did not initiate active metabolism and could conserve the energy for long-term survival. This observation suggests that integration of carbon, nitrogen and phosphorus sensing is imperfect in V. cholerae and it cannot down-regulate the metabolism during phosphorous limitation. The carbon and nitrogen could prime the cells to accelerate the rate of metabolism, irrespective of the presence of phosphorous, thereby creating an energetically unfavorable situation. The lack of crucial component phosphorus fails to activate a stringent response that results in increased futile cycling of nutrients, loss of ATP and cell death. The Vibrio genus was found to be less efficient in surviving phosphate starvation than E. coli and S. Typhimurium. The two-component system CreC (a response regulator of phosphorous) is absent in V. cholerae and may be responsible for the lack of stringent response to phosphorus starvation.

Campylobacter jejuniis is the most common cause of bacterial gastroenteritis worldwide. It is often considered a ‘commensal’ in chickens where it rapidly colonises the caecum of young chicks and is present in around 80 % of farmed poultry. C. jejuni has a small genome and most strains are unable to transport or metabolize glucose. Instead they use amino acids, TCA cycle intermediates and short chain fatty acids as energy and carbon sources. Recently, a WGS project identified an unusual group of C. jejuni (RG-2 group) within a bank of C. jejuni strains isolated from farm associated Norway rats. These strains had acquired an entire locus (glc) of seven genes which enable uptake and metabolism of glucose via the Entner Doudoroff (ED) pathway. This project is addressing the impact of these genes on metabolism and niche survival of these strains. Campylobacter are microaerophilic bacteria. Good growth of C. jejuni NCTC 11168 and of aglcnegative Norway rat C. jejuni isolate was recorded in the presence of 5 % O2up to 13 %in a Whitley M35 workstation. In contrast, two glcpositive strains of C. jejuni, Dg275 and Dg95, exhibited the unusual ability to grow well in an atmosphere of 16 % oxygen, as monitored by c.f.u. and OD600. Current studies are also focusing on the contribution of glucose utilization via the ED pathway to influence survival and growth in oxygen.

The survival of enteric pathogens in the multi-nutrient environment involves interaction with other organisms. The fact that competition for nutrient resources has shaped the networking of metabolic pathways is well known. The cAMP receptor protein i.e. CRP is a keystone regulatory protein, connecting various metabolic pathways. Our aim was to study the importance of CRP in nutrient uptake and utilization in Salmonella Typhimurium under intra-species and inter-species nutritive competition. The crp gene knockout (Δcrp) was co-cultured with the wild-type or other pathogens. The Δcrp failed to compete with the wild-type Salmonella Typhimurium, Escherichia coli, Vibrio cholerae and Staphylococcus aureus in nutrient intensive media. However, the survival of the co-cultured Δcrp was unaffected in nutrient-poor media. These results suggest that CRP is necessary for the effective acquisition of readily available nutrients as found in rich media in co-culture. The role of released antimicrobials or surface proteins of the co-cultured strains was also overruled by culturing the mutant in the supernatant of these organisms and separating the cultures in the same media respectively. The co-cultured Δcrp showed an enhanced survival when overall metabolism was reduced with low temperature and antibiotics like chloramphenicol. A circumstantial evidence that CRP manages the global and limits the futile metabolism is provided by this study. The absence of CRP doesn’t affect the survival of the standalone culture. However, the role of this protein becomes obvious in a bacterial community setup. Therefore, CRP is crucial for Salmonella to survive in an intestinal and external environment.

For bacteria, mechanisms of resistance to redox stress are utilised to increase survival. Although B. henselae has the ability to resist redox stress, its genome sequence is characterised by a paucity of genes responsible for redox-stress resistance, particularly hydrogen peroxide degradation systems. However, our results show a surprisingly high resistance to peroxides, given the lack of peroxide disposal systems. To determine how B. henselae achieves resistance to H₂O₂ stress, the potential role of MbfA (membrane-bound bacterioferritin), which in other α-Proteobacteria is believed to function as an iron exporter, was investigated. The results show that B. henselae has the ability to export iron and that this export activity is promoted by H₂O₂as export was inhibited by exogenous catalase and anaerobiosis. The form of iron exported was largely ferric. The impact of the iron export process on the resistance to, and degradation of, H₂O₂, by B. henselae was determined and the results showed that B. henselae mediates a rapid consumption of exogenously supplied H₂O₂. This degradation was entirely inhibited when iron chelators were included along with the H₂O₂. The resistance of B. henselae to NO was also tested since NO is generated by phagocytic host cells along with H₂O₂, and is suggested to potentiate the toxicity of H₂O₂ towards engulfed bacteria through inhibition of haem-dependent catalases and alkylhydroperoxidases. Our results suggest that NO does not cause a marked increase in H₂O₂ toxicity for B. henselae, in contrast to E. coli (a haem-catalayse/peroxidase dependent bacterium).

Streptomyces coelicolor is a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosis as an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Heat kill assays show that the deletion of the mce operon result in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. The spores of a Δmce mutant also exhibit a precocious germination phenotype seen on SEM images confirmed with germination assays.

The contemplation of present research work to get over scantiness of amino acid by amelioration ofmicroorganism. As a matter of fact the use of chemical and physical factors symbolize a new era in the excerption of microorganism for proficient of producing a desired product. In this workbiosynthetic pathway of valine and their regulation was observed in Bacillus cereus, Corynebacterium and Pseudomonas fluoresence. The contrivance of operation of ems mutants was analyze in different fermentation media at 24, 48, 72 and 96 h incubation under optimum condition by acidic ninhydrin method. Optical density was recorded by spectrophotometer 470 nm for Valine. The effects of carbon and nitrogen sources and growth factors on the production of valine were studied. In this studies mutant evince that mutation confabulate upon the bacteria the ability to produce valine many times more than its parental strain within the same span of incubation period. The objective of this work to step up headway to search unconventional sources to overcome nutritional exhortation of valine.

The interaction between transcription factors, promoter elements and RNA polymerase is crucial to bacterial adaptation to physical and chemical changes in the environment. Transcription factor discovery has strongly relied on the isolation of regulatory mutants followed by biochemical confirmation. However, highly pleiotropic regulatory mutations can often be deleterious, unstable and difficult to select, or could directly or indirectly affect gene expression at multiple levels. Here, we describe a biochemical approach to identify trans-acting regulatory proteins independent of their cellular function that could be used as an alternative to genetic screens. The method consists in (i) incubating bacterial lysates with an immobilized DNA encompassing a promoter as defined by RNA-Seq data (ii) pull down of bound proteins and (iii) liquid chromatography tandem mass spectrometry (LC/MS/MS). We tested the usefulness of this approach by identifying proteins binding to the Vibrio cholerae rpoS promoter that drives the expression of the general stress response regulator RpoS. The approach identified several proteins binding to the rpoS promoter that included the factor for inversion stimulation (FIS), and the master quorum sensing regulator HapR. Binding of both purified proteins to the rpoS promoter was confirmed by electrophoresis mobility shift assays. The role of fis and hapR on rpoS expression was examined in strains containing a chromosomally-integrated rpoS-lacZ fusion. Deletion of fis had little effect while HapR appeared to enhance rpoS expression.

A comprehensive understanding of bacteria phenotypes requires tools that are able to characterise structure and function across multiple length scales, from communities and individual cells down to single molecules. Multimodal sensing combines multiple transduction technologies in parallel to probe different properties simultaneously, and thereby increase the range of measurable interactions, the amount of information that can be extracted, and improve detection accuracy. Electrophotonics is a multimodal sensing approach that combines electrochemical and photonic techniques in a single, integrated device that provides enhanced quantitative measurements of chemical reactions. We present a new electrophotonic device based on a Si3N4 guided mode resonant (GMR) structure with an integrated indium tin oxide (ITO) electrode. The GMR structure is sensitive to refractive index changes at the sensor surface, enabling label free, real time detection of biomolecules, microorganisms and imaging of molecular interactions with micrometre-scale resolution to provide spatial information about surface binding interactions. The ITO electrode has been shown to be compatible with voltammetry-based techniques for interrogating redox behaviour along with electrochemical impedance spectroscopy. We demonstrate the wide range of microbiological applications of electrophotonic technology including the characterisation of redox active protein electron transfer and surface adsorption, bacterial adherence and growth on chemically functionalised surfaces, and label free parallel detection of clinically relevant biomarkers. We believe that the multimodal measurements with this novel technology can provide new approaches to investigate and understand microbial biology.

D-serine is an amino acid that has become a focus in recent years due to its unique role in many biological processes. It is a host metabolite in humans with diverse roles in neurotransmission and signalling. Previous work in our group showed that D-serine can play a critical role in controlling expression of pathogenic virulence factors in bacteria, specifically Escherichia coli, as well as impacting microbial community composition through niche specificity. In enterohaemorrhagic E. coli (EHEC), the presence of D-serine results in down regulation of the type 3 secretion system (T3SS), resulting in inability to colonise. In contrast, uropathogenic E. coli (UPEC) can catabolise this metabolite and colonise the bladder where D-serine is present in high concentrations, leading to a urinary tract infections (UTI). Hence, in different pathotypes, D-serine can act as a positive (treat) or negative (trick) environmental stimulus. UPEC and neonatal meningitis E. coli (NMEC) strains often carry the DsdXCA operon which allows for the metabolism of D-serine. This locus is responsible for the detoxification of D-serine, allowing for tolerance of D-serine as a carbon source. My work has focused on understanding the role of D-serine as a signal for gene expression in UPEC and NMEC through the action of the regulator, DsdC. Using global approaches I have characterised the binding sites of DsdC across the chromosome revealing new insights into how this protein contributes to UPEC and NMEC pathogenesis. The work is important as it helps us understand how specific pathogens sense their environment and cause disease.

The dominance and pathogenicity of Helicobacter pylori in the gastric niche is eminent. Urease test is used to diagnose the infection of H. pylori. However, nowadays several reports indicated the emergence of organisms other than H. pylori (Non H. pylori bacteria– NHPs). We have reported urease positive, Ochrobactrum intermedium from the non-ulcer dyspeptic individual. Therefore such urease positive bacteria raise a question on the reliability of urease test. Since 2005, nearly 35 cases are reported for the presence of Ochrobactrum spp. from clinical specimens. Their high level of resistance to antibiotics and phylogenetic relationship to anthropozoonotic pathogen, Brucella makes them a probable human pathogen. Therefore, it is necessary to study their prevalence, survival mechanism and function in the human stomach. To check their pervasiveness in the Indian population, 218 urease positive gastric biopsy samples were processed and 62 Ochrobactrum spp. isolates were identified with and without H. pylori. Population study of Ochrobactrum spp. was also done using multilocus sequence typing (MLST) which revealed 45 sequence type and clonal population. As the prevalence of Ochrobactrum spp. is approx. 30 %, their survival mechanism was studied under in vitro gastric conditions (acidic pH, urea, microaerophilic environment), using microarray and RNA seq methods. The differential gene expression analysis showed 2 different acid resistance mechanism, i.e. Urease dependent acid resistance mechanism like H. pylori, and Amino acid dependent acid resistance mechanism. Our finding necessitates further detailed investigation of such NHPs in gastric environment and role of such bacteria in gastric niche and warrants further refinement of urease based diagnosis.

Tuberculosis (TB) is the leading cause of global morbidity and mortality caused by an infectious disease, with over 10 million new cases emerging in 2017. This global emergency is exacerbated by the emergence of multi-drug and extensively-drug resistant TB, therefore new drugs and new drug targets are urgently required. From a whole-cell phenotypic screen we identified a series of nitrogen-containing heterocyclic compounds that elicit potent anti-mycobacterial activity with MIC values <10 µM against Mycobacterium tuberculosis. Interestingly, this series of compounds demonstrate no detectable drug resistance in mycobacteria. Mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with late-stage mycolic acid biosynthesis. In addition, these compounds exhibit a suitable toxicological and PK/PD profile that paves the way for further development as an anti-TB chemotherapy.

Pediocin PA −1 is class IIa bacteriocin that displays efficient antimicrobial activity against pathogenic Listeriaspp. This bacteriocin is known to lose activity during long periods of storage especially at non optimal pH, thus reducing its usefulness for the pharmaceutical and food industries. Loss of activity has been attributed to oxidation of the methionine residue at position 31, however, replacement of this residue by leucine results in a peptide with activity equivalent to that of the native peptide. In this work, the heterologous expression of the structural (with Met31 to Leu substitution), accessory and transport genes from pediocin PA-1 operon was carried out in Escherichia coli TunerTM (DE3) cells. The sequences of all genes were redesigned using codon bias for the host and were cloned into an expression vector that allows control of plasmid copy number. The heterologous expression of pediocin Met31Leu was optimized for temperature, induction time, IPTG concentration and plasmid copy number and was evaluated via antimicrobial activity assays against Listeria innocua DPC3572. Maximum activity (2560 AU mL−1) was achieved using low plasmid copy number and 6 h of induction at 37 °C with 1 mM of IPTG. Recombinant pediocin PA-1M31L was successfully purified in 5 steps (>95 % purity) as confirmed by mass spectrometry (4606.27 Da) with a yield of 0.725 mg per liter of culture. This variant showed a similar spectrum of activity to the native pediocin PA-1 and is an interesting alternative for industrial applications due to its greater stability.

Dimer formation is a serious threat to the stable maintenance of ColE1-like plasmids. Dimers form infrequently by homologous recombination but accumulate rapidly by having two origins of replication. This results in elevated plasmid loss and a reduction in host cell growth rate. Plasmid dimers are resolved to monomers by the XerCD recombinase plus accessory proteins ArgR and PepA, acting at the cer recombination site. The circular chromosome of E. coli also forms dimers infrequently, and consequent failure of chromosome partition results in filamentation, SOS induction, and failure of cell division. Site-specific recombination is required for dimer resolution during cell division in a process facilitated by XerCD acting at the dif (deletion induced filamentation) site near the E. coli chromosome terminus. ArgR and PepA accessory proteins are nonessential, but the septum-associated protein FtsK is necessary for dimer segregation, suggesting the XerCD/difcomplex interacts with division septums. Our preliminary work had revealed homology between cer and a 170 bp chromosomal site (tcs, terminal region cer-like site) 1.2 min from dif. The tcs site and surrounding region was cloned into plasmids, dimer plasmids were purified and then assayed for tcs-mediated recombination. Our results demonstrate that a construct with a 500 bp tcs insert supports XerCD-mediated recombination, whereas smaller constructs were recombination-deficient. The absence of plasmid monomerization in mutant strains indicates that ArgR and PepA are required for recombination. Additionally, the tcs knockout strain displayed moderate filamentation of cells. Given the similarities between cer, tcs, and dif, these results suggest that tcs could facilitate chromosome dimer resolution.

While transcriptional reprogramming is perhaps the most well understood form of controlling gene expression in response to nitrogen starvation in bacteria, how post-transcriptional regulation (PTR) of gene expression contributes to this adaptive response remains elusive. Small regulatory RNAs (sRNAs) are the major post-transcriptional regulators of gene expression in bacteria. They regulate gene expression by base pairing to target mRNAs, leading to enhanced translation or inhibition of translation and/or alteration of mRNA stability. To form productive interactions with target mRNAs, most sRNAs require an RNA chaperone. In many bacteria of diverse lineages, the RNA chaperone Hfq plays a central and integral role in the PTR of gene expression by stabilising sRNAs and promoting their interactions with cognate mRNAs. Comparative analysis of the transcriptomes of Escherichia coli at different stages of nitrogen starvation reveal that levels of sRNA vary throughout starvation. We used Hfq as a surrogate to study sRNA-mediated PTR of gene expression during sustained nitrogen starvation. Our results indicate that sRNAs-mediated PTR of gene expression plays a major role in the adaptive response to sustained nitrogen starvation. Intriguingly, using single-molecule PALM, we reveal that Hfq is involved in the formation of intracellular structures which functionally might resemble processing (P) bodies found in eukaryotic cells involved in mRNA turnover.

Human nasal colonization with Staphylococcus aureus sets the stage for subsequent systemic infection. The mechanisms responsible for colonization are not fully understood. This study characterizes S. aureus adhesion to nasal mucosa in vitro and observes the interaction of S. aureus with mucin. S. aureus showed significantly higher binding to mucin during stationary phase in comparison to log-phase cells. Adherence of S. aureus srtA mutant to mucin was not significantly different from wild-type, thus Srt A shows no influence on S. aureus interaction with mucin. Adherence to mucin was saturable in a dose- and time-dependent fashion. Biotin–labelled mucin bound to surface protein (55 kDa) of cell wall extracted S. aureus which is encoded by the sbi gene. These data suggest that adherence factors are present on the surface of S. aureus such as sbi. Purified recombinant Sbi was prepared and the mucin binding capacity of the protein was tested by ELISA. In order to determine the function of specific domains of Sbi in adhesion, Sbi constructs with, without the IgG-domain and with B2-glycoprotein domain were expressed on the surface of E. coli BL21(DE3). The expression of Sbi with the B2-glycoprotein domain on the surface adhesion, emphasizing the role of Sbi in mucin adhesion. A profound binding effect was observed with Sbi incubated in wells coated with host mucin. Therefore, it is proposed to investigate a novel interaction of S. aureus to host mucin in order to control S. aureus nasal colonization.

Actinobacteria have a protein O-glycosylation system that resembles eukaryotic protein O-mannosylation. Both M. tuberculosis and S. coelicolor have growth retarded phenotypes when protein-O-mannosyl transferase (Pmt), which transfers mannose from polyprenol phosphate mannose to a target protein, is absent. Moreover, S. coelicolor pmt- mutants are resistant to φC31 phage infection and have increased susceptibility to vancomycin and multiple b-lactams. S. coelicolor strains that lack polyprenol phosphate mannose synthase (Ppm1), which transfers mannose from GDP-mannose to polyprenol phosphate, are even more susceptible to antibiotics and a ppm1- mutant in M. tuberculosis is lethal. Pmt and Ppm1 are therefore possible new targets for the isolation of antimicrobials to be used against M. tuberculosis. Our aim is to further understand the structure and function of these enzymes. Sequence alignments and structural bioinformatics were used for S. coelicolor Ppm1 and Pmt to identify site-directed mutagenesis targets. Mutant alleles were introduced into ppm1- (DT3017) or pmt- (DT2008) S. coelicolor strains using conjugative integrative plasmids and scored for their ability to complement phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-two highly conserved Pmt residues were each changed to alanine and six mutants failed to complement DT2008, indicating essentiality. Modelling these six residues indicated that five are positioned close to the predicted catalytic DE motif. For Ppm1, ten mutant alleles were tested and eight were essential for DT3017 complementation, with four residues positioned close to the predicted catalytic DXD motif. Whilst some of the mutations were predicted to impair catalytic activity, others may have affected localisation or substrate binding.

We previously identified the diversity of antimicrobial activity in nasal microbiomes that correlates with presence and absence of Staphylococcus aureus. The major competitor of skin surfaces is Staphylococcus epidermidis that can express diverse antimicrobial activities. Ten-fold less frequently found in skin and nasal microbiomes, Staphylococcus hominis is relatively unstudied. The aims of the research were to develop insights into the factors of S. hominis that contribute to the dynamics of competition with these major skin colonising staphylococci. One prominent inhibitory strain, S. hominis was selected to culture with both S. aureus SH1000 and S. epidermidis with aim to use genome sequencing to reveal loci contributing to competition. S. aureus evolved during the competition of S. epidermidis and inhibitor-producing S. hominis to reveal two discrete phenotypes. Non-pigmented clone expressed less alpha-haemolysin and had a SNP in agr Cencoding the receptor of the Agr quorum-sensing system. More Pigmented clone had a SNP in sig Bencoding the accessory sigma factor required for expression of the staphyloxanthin. Distinctively, competition of S. aureus SH1000 with S. epidermidis revealed evolution of S. aureus that corresponded with a SNP in the lytSgene of the LytSR two-component system controlling murein hydrolase activity and autolysis. Pacbio genome sequencing followed by use of AntiSMASH revealed the S. epidermidis inhibitory strain, which enabled persistence of S. hominis, encoded the lantibiotic gallidermin biosynthesis operon on a 39 kb plasmid. Gallidermin decreased S. aureus survival to competitor S. epidermidis, and an increased ability of S. hominis to maintain its population size during evolution experiments, which supported the dynamic relationship of the three staphylococci. Future studies will unravel explanations for the contributions the identified loci make to multi-species competition.

Bacterial biofilms are highly complex communities, composed of highly structured extracellular polymers and subpopulations of differentiated cells, such as persisters. These contribute to the resistance of bacterial biofilms to antibiotics, creating a significant issue in the treatment of infections and resulting in elevated levels of mortality and morbidity. Here, we use a microfluidic system coupled with time-lapse microscopy and fluorescent dyes for exopolysaccharide (EPS) and extracellular DNA (eDNA) to investigate how the architecture of the growing biofilm is ordered. We show that in Pseudomonas aeruginosa EPS is deposited first in the initial stages of microcolony development, but that eDNA then acts as a leading edge for further microcolony expansion. We explore how this assembly is perturbed by the introduction of different antimicrobial agents. Further, working with partners in the European Association of National Metrology Institutes (EURAMET) we are developing cross platform methods for the label-free localisation of antimicrobial agents and bacterial components within the biofilm. These platforms include 3D OrbiSIMS (secondary ion mass spectrometry) and Raman spectroscopy. Here we demonstrate the localisation of key biofilm components such as Pseudomonas Quinolone Signal (PQS) molecules in a 3D chemical map of the biofilm.

Gut eukaryome refers to the collection of fungi and protists in the gut. Until recently, all eukaryotic gut microbes were considered parasites and subject to elimination. Though this is true in some instances, critical evaluation of the literature reveals that most microbial eukaryotes are harmless often colonizing the human gut for long periods of time. Evidence is accumulating that the eukaryome plays important ecological roles in gut communities, as well as, in health and disease of the host. Nonetheless, systematic examination aiming to obtain baseline information of their prevalence and diversity in human populations is lagging. To address this knowledge gap, we collected fecal samples from a population of adults residing in north Thailand (n=211), who showed no gastrointestinal (GI) symptoms and had no history of GI diseases. We then examined the prevalence and diversity of two commonly found eukaryotic genera: the stramenopile Blastocystis (Blastocystis spp.) and the yeast Candida (Candida tropicalis and Candida albicans). Twenty three percent of individuals were positive for Blastocystis. Their sequences grouped in six of the nine clades that colonize humans. Twelve percent of the study population was positive for Candida, 4 % for C. albicans and 8 % for C. tropicalis, while concurrent colonization was also noted in some individuals. Eukaryotic bacterial interactions, as well as, interplay with diet and body mass index are also discussed. This is the first study providing data on the eukaryome of Thai populations and evidence that microeukaryotes traditionally considered as pathogenic asymptomatically colonize the gut of healthy humans.

Worldwide, hundreds of thousands of healthcare acquired infections (HAIs) are reported each year. Contamination of hospitals is a source of, and allows dissemination of HAIs. In healthcare settings one of the major vectors of contamination is healthcare workers’ uniforms. As surfaces become contaminated, bacteria can then be contacted by patients or staff who may indirectly spread bacteria to patients. Direct and indirect spread of bacteria could result in infection of patients and increased infection rates. Further consequences include increased levels of antibiotic use and costs. A pilot study was conducted at Antrim Area Hospital, Northern Health and Social Care Trust. 100 pre-shift and 100 post-shift healthcare workers’ uniforms were assessed for Staphylococcus aureus and Enterococcus spp. isolates. We found increased levels of antibiotic resistant S. aureus and Enterococcus spp. contamination on post-shift uniforms compared to zero to minimal contamination of pre-shift uniforms. A biobank of isolates was subsequently characterised for antibiotic sensitivity using European Union Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines – 51 % of S. aureus isolates were classed multi-drug resistant. Genomic diversity was assessed using Random Amplification of Polymorphic DNA (RAPD) – high levels of similarity was found amongst isolates. As one means of reducing uniform bioburden, we conducted analysis of a novel surface-active organisilane disinfectant named Goldshield (GS). GS was marketed as a long lasting antimicrobial prevent (re)contamination. GS technology displayed bactericidal, sporicidal and anti-biofilm properties in laboratory testing providing rationale for an intervention where GS could be incorporated into hospital laundry and assessed for potential use in infection control.

Diarrhoeal disease is a major public health concern, with over 500 000 childhood deaths recorded every year, (www.who.int). The leading cause of infantile diarrhoea is pathogenic Escherichia coli (EPEC), characterised by watery diarrhoea which varies in levels of severity. In recent studies a growing incidence of asymptomatic EPEC carriage has been recognised in Asia and the Middle East, suggesting the possibility that communities may act as a reservoir for this microorganism, (Kader, 2009; Alikhani, Mirsalehian and Aslani, 2018). Using a combination of molecular biology and bioinformatic analysis, we aim to characterise asymptomatic strains from these regions, determine their key virulence factors and find new ways of combatting this pathotype. At present we have developed a diagnostic multiplex PCR which incorporates known virulence genes of diarrhoeagenic E. coli pathotypes for quick and simple detection of EPEC. Additionally, we have optimised an infection assay using HeLa cells to phenotypically characterise typical and atypical colonisation and also identified bacteriocins capable of killing EPEC.

The presence of fungal species on the surface skin and hair is a known finding in many mammalian species and humans are no exception. Superficial fungal infections are sometimes a chronic and recurring condition that affects approximately 10–20 % of the world’s population. However, most species that are isolated from human tend to occur as co-existing flora. This study was conducted to determine the diversity of fungal species isolated from the hair and nails of workers in the central region of Saudi Arabia where there are not many observational studies on the mycological species. Male workers from Riyadh, Saudi Arabia were recruited for this study and samples were obtained from their nails and hair for mycological analysis which was done using Saboraud’s agar and sterile wet soil. Fungal isolates were examined microscopically. Twenty four hair samples yielded a total of 26 species from 19 fungal genera. Chaetomium globosum was the most commonly isolated fungal species followed by Emericella nidulans, Cochliobolus neergaardii and Penicillium oxalicum. Three fungal species were isolated from nail samples, namely, Alternaria alternata, Aureobasidium pullulans and Penicillium chrysogenum. Most of the isolated fungal species (17 of the 26 or 65.38 % of the isolated fungal species). Most of our isolated fungal species have not been thoroughly characterised nor morphologically classified.

Perkinsus species are important marine parasites of molluscs including mussels and oysters with substantial commercial and environmental impact. Perkinsus forms a sister lineage to the apicomplexan parasites (e.g. malaria agent, Plasmodium) and share similar invasion-related structures with these better studied pathogens. However, much less is known of the transmission and invasion biology of Perkinsus spp. We are developing genetic tools to study these parasites, and Perkinsus is emerging as an experimental model for marine parasites. The life cycle of Perkinsus species start with motile zoospores that are ingested by the host via filtration of sea water. After infection of the mollusc, these cells develop into a replicating non-motile vegetative form of the parasite, the trophozoite. Upon host death or morbidity, the parasites are released back into the water column where they differentiate into zoosporangia that release up to 100 motile zoospores. In vitro, Perkinsus species can be maintained as trophozoites in an axenic sea water-based media, and this is the best studied form of the parasite. However, Perkinsus olseni, can be triggered to sporulate in vitro using Ray’s fluid thioglycollate media, and we seek to better study this phase of the lifecycle that is essential for disease spread. To do this, we are determining stage-specific gene expression, during the transformation from trophozoites to zoospores, by RNA-Seq. This will both identify genes that are specific to this growth stage, and also identify zoospore-specific promoters that can be used in experimental manipulation and study of this poorly known cell stage.

Mastitis is a common disease in dairy cattle. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland. Most of the isolates analyzed demonstrated an ability to produce biofilm (over 70 %). Virulence genes were searched for in S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33 %), cylE (78 %), bca (37 %), and cfb (100 %). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes were Ia (n=8) and II (n=8), in addition to some strains that were not classified in any of the groups (n=6). Out of the eight selected genes for S. uberis only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76 % and 86 % of the strains, respectively. The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.

Colibacillosis caused by Avian Pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality and carcass condemnation to the poultry industry worldwide resulting in significant economic losses. We assessed a multiplex PCR for classifying diagnostic APEC and characterized these isolates using gene profile analysis.48 E. coli isolates collected between August and October 2018 through the Poultry Disease Research Center (PDRC) Diagnostic Laboratory were analyzed. Isolates were screened using multiplex PCR targeting genes associated with APEC chromosomal and plasmid virulence. Isolates were assessed for relationship between gene profile and host tissue of origin. Overall, isolates met the criteria for definition as well-developed pathogens with more than 90 % of isolates positive for the genes iroN, ompTp and hlyF; 78 % were positive for aerJ and 67 % for iss. A significantly lower prevalence was observed for cvaC, etsB, ireA and papC (range 5–36 %). When overall gene prevalence was examined for tissue of isolation, we found that APEC from the ovary, bone marrow, pericardium and lung had higher average numbers of genes compared to isolates recovered from skin and yolk sac. Genes associated with the ColV virulence plasmid (iss, iroN, hlyF and ompTp) were detected in 43 of 48 isolates (89.5%) further confirming the ColV plasmid is the defining trait of the APEC subpathotype. The use of a multiplex panel to screen for APEC has shown good correlation with pathogenesis, and tissue source and correlates well with invasive strains. Path panel diagnostics is available through PDRC, providing significant value to APEC screening.

Uneven flock growth (UFG) occurs when a broiler flock exhibits a wide range of bodyweights at slaughter. Automated chicken processing plants operate on an average bodyweight for the flock being processed so UFG can cause disruption to the processing flow that requires manual remediation. It is known that enteric virus infections of young broiler flocks lead to varying degrees of growth restriction that range from runts at hatch and severe early stunting, which are generally culled, to poor flock performance and UFG. In order to investigate the make-up of communities of enteric viruses (virome) associated with poor growth and also their infection timings and persistence in broiler flocks, a longitudinal survey of 7 commercial broiler flocks of varying performance over 3 successive broiler crops was undertaken with gut and faecal samples collected from 50 birds at each of 12 timepoints from pre-hatch to slaughter. The samples were pooled for each timepoint, then processed to enrich for viruses by removing host cells and bacteria. The RNA was extracted from each timepoint sample, amplified by whole transcriptome amplification followed by whole genome library preparation for the Illumina MiSeq platform and libraries multiplexed. Bioinformatics analysis used ViromeScan which facilitates metagenomic studies of quality-trimmed sequencing reads. The presence of diverse viral families was seen including the known major enteric viruses of the astrovirus, picornavirus, reovirus and parvovirus families. A greater diversity of viruses was observed in the flocks of poorer performance than in those flocks of good performance.

Dothistroma needle blight caused by D. septosporum has emerged in the British Isles as a major threat to Corsican pine, lodgepole pine and Scots pine. There is increasing evidence that mycoviruses can reduce the growth and pathogenicity of fungal plant pathogens. The aim of the present study is to characterise a double-stranded RNA virus found in D. septosporum and investigatefor putative hypovirulence, a common feature noted for mycoviruses, which might be used for biological control to invasion by more aggressive strains of the fungus. To this endthe viral genome was cloned and sequenced revealing four genomic segments, each one containing a single open reading frame (ORF) flanked by 5’ and 3’ untranslated regions. The ORFs encode the RNA-dependent RNA polymerase, the capsid protein, a protein of unknown function and a putative protease, respectively. Phylogenetic analysis of the sequences obtained revealed their similarity to members of the established family Chrysoviridae, genus Alphachrysovirus, which are encapsidated in isometric particles and are known to elicit hypovirulence in their hosts. Subsequently, virus-free and virus-infected isogenic lines were generated to determine any effects of the mycovirus on fungal fitness and pathogenicity. More specifically, the virus-infected isolate is currently being assessed in comparison to the virus-free one in terms of radial growth in solid culture, biomass in liquid culture, pathogenicity in pine trees and production of the mycotoxin dothistromin. In conclusion, this study reports the first mycovirus ever found in D. septosporum.

Clostridioides difficile, formerly known as Clostridium difficile, is a pathogen of increasing agricultural and clinical significance. This Gram-positive, anaerobic, toxigenic bacterium produces extremely robust spores which are highly resistant to environmental pressures, allowing this organism to persist in the environment for extended periods. C. difficile is capable of infecting both animals and humans. As a commensal organism it does not typically pose a threat to its host unless antibiotic treatment disrupts the normal gut flora. However following a decline in microbial diversity, C. difficile opportunistically colonises the host gut and proliferates, thereby causing C. difficile infection (CDI). CDI is transmitted via the faecal-oral route with spores surviving transit through the gastrointestinal tract to the gut. Symptoms of CDI range from mild diarrhoea to pseudomembranous colitis (PMC), toxic megacolon and death. C. difficile ribotypes present in farm animals have been found in workers tending to them, suggesting a potential zoonotic transmission. Sporulation is intrinsic to C. difficile’s transmissibility and the spores are highly resilient. Previous joint work at both the Queen’s University Belfast and Royal Victoria Hospital (Belfast) identified a non-sporulating variant of ribotype 078, the most prevalent ribotype infecting humans in Northern Ireland. Investigations into the prevalence of the non-sporulating variant in a range of animal and clinical derived ribotype 078 isolates are currently ongoing. Additionally, the presence of the non-sporulating variant in other toxigenic clades of C. difficile is undergoing assessment.

Brucella organisms are Gram-negative, intracellular facultative extracellular bacteria that infect a variety of animals. On March 2018, a pregnant dwarf sperm whale (Kogia sima), stranded and aborted on Herradura beach at the Pacific coast of Costa Rica. K. sima is a criptic species; very little is known of its biology and worldwide distribution, however it is still hunted in Asia. Brucella sp. was recovered from multiple tissues of the female and the calf, and examined by biochemical tests, MLVA-16, brucellader and HRM real time. The isolates were used for whole genome sequencing and the reads were aligned to Brucella abortus 9–941 as the reference, alltogether with other Brucella species. A total of 27 365 variable sites were extracted and a phylogenetic reconstruction by maximum likelihood was produced. The phylogenetic tree revealed that the K. sima isolates are related to Brucella sp. F5/99, a singular strain recovered on 1992 from a bottlenose dolphin captive in California, classified as sequence type (ST) 27 by multi-locus sequence type. This ST27 was described in Brucella isolates with zoonotic capacity and therefore transmission to humans from Peru and New Zealand. This is the first report of Brucella ST27 recovered from a host of the Eastern Tropical Pacific and of Brucella infection in a dwarf sperm whale. Our results shows that the range of marine mammals infected by Brucella sp. is wider than our current knowledge, and that biosafety measures should be increased when handling the stranded mammals, as the zoonotic transmission is of major risk.

Copper is an essential metal in both eukaryotes and prokaryotes, however excess levels are toxic. Bacteria have developed mechanisms, such as efflux and sequestration, to counteract these toxic effects. Significantly, copper has been shown to be important in host innate immunity as an antibacterial mechanism against invading pathogens, via active transport of copper into the phagosome. Worryingly, there has been a global emergence of S. aureus strains with increased antibiotic resistance (e.g community-acquired methicillin resistant S. aureus (CA-MRSA)), which unlike typical S. aureus, can infect healthy humans with no previous exposure to healthcare situations. These isolates show increased resistance to innate immunity and reduced clearance from healthy airways compared to other clinical isolates. Recently, we identified a novel horizontally transferred copper resistance locus, copXL, in CA-MRSA which is in addition to the core copper homeostasis operon (copAZ) found in all S. aureus and is not present in established S. aureus human lineages. copXencodes a copper efflux transporter and copLis predicted to encode a lipoprotein of unknown function. This operon confers resistance to extremely high concentrations of copper compared to other S. aureus and, notably, is important for survival within intracellular macrophages. The recent evolution and success of USA300 may be due to possession of these additional copper resistance genes, enhancing bacterial fitness through increased resistance to copper-dependent bactericidal innate immunity. The function of CopL in S. aureus macrophage survival and copper hyper-resistance is currently being investigated to combat this highly effective copper resistance mechanism and spread of these highly virulent pathogens.

The human pathogen Helicobacter pylori colonises approximately half of the global population and infectioncan lead to a range of gastric diseases. The temperate bacteriophages in H. pylorihave been poorly characterised, most likely due to a very high number and strain-to-strain variability of restriction modification (RM) systems, which can be easily more than 20 in any strain. This work aims to study the prevalence of bacteriophages and RM systems in over 460 strains of H. pyloriisolated from 184 gastric samples from asymptomatic subjects. H. pyloriprophages were identified using the PHASTER tool. The gold standard RM systems were downloaded from Rebase and the RM genes were identified in the 460 genomes using Blast+. The analysis for phage genomes showed 57 intact bacteriophages, ranging from 12 to 30 Kb in length, in 25 subjects (14 %). Approximately half of the bacteriophages were shown to have integrated in the Lipid A biosynthesis gene lpxD. Using 107 RM system genes, we built a presence/absence matrix of the genes in all our H. pylori genomes, which was ordered according to a maximum likelihood gene presence/absence tree generated by FastTree. This clustering revealed the presence of multiple pairs of strains, one strain carrying an intact prophage whereas the other is lacking a prophage but containing the same RM systems, potentially allowing for a prophage donor and recipient pair. The availability of this panel of donor and recipient strain pairs is an ideal starting point to study the molecular biology of bacteriophages in H. pylori.

Burkholderia cenocepacia, an opportunistic bacterium causing chronic respiratory infections in patients with cystic fibrosis, produces the bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures antibiotics outside bacterial cells, providing a novel extracellular mechanism of antimicrobial resistance. Sublethal concentrations of bactericidal antibiotics (e.g. polymyxin B, norfloxacin) and superoxide ion stress inducers (e.g. paraquat) stimulate transcription of bcnA, suggesting BcnA may also play a role in oxidative stress responses. bcnA gene homologues can be genetically linked to an upstream gene encoding a predicted membrane cytochrome b-561 protein, which we have named bcoA (bcnA cytochrome oxidase-associated gene). RT-PCR showed that both bcnA and bcoA are co-transcribed in B. cenocepacia. We hypothesize that BcnA and BcoA may be components of an unrecognized pathway to protect B. cenocepacia and other bacteria from membrane lipid-derived peroxidation damage. Here, we show that bcnA and bcoA deletion mutants display enhanced membrane lipid peroxidation and fail to survive under conditions that stimulate peroxidative stress (e.g. cold stress and salt stress). Compared to wild type, the levels of the lipid peroxidation biomarker malondialdehyde are also increased in the mutants upon exposure to near-lethal concentrations of polymyxin B and norfloxacin. No differences in bacterial survival were detected in parental and mutants under anaerobiosis. These findings indicate that BcnA and BcoA participate in a previously unrecognised peroxidation detoxification pathway that likely protects the outer membrane lipid bilayer under extreme stress conditions such as antibiotic killing.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can cause haemorrhagic diarrhoea and potentially fatal renal failure in humans. Ruminants are considered the primary reservoir for human infection. Studies investigating the response of cattle to colonization generally focus on humoral immunity, leaving the role of cellular immunity unclear. These bacteria colonise their host by tight attachment to the epithelium, using a type three secretion system to inject a cocktail of effectors into the host cell. Injected effectors manipulate the innate response in several ways to promote bacterial persistence. Transcriptional profiling of responses at the terminal rectum, the primary site of colonisation in cattle, reveals a bias towards a T-helper type 1 response, indicating that cellular immunity may be involved in bacterial clearance. Mathematical modelling also indicates that injected effectors have a reduced human MHC-I epitope density, whilst structural bacterial proteins do not. This implies that host cellular immune responses target injected effectors and have exerted selective pressure on their evolution. My on-going research is examining the expression of MHC-I on the surface of cultured bovine epithelial cells following infection with E. coli O157. Initial results demonstrate a decrease in MHC-I surface expression during EHEC O157 colonisation after three hours. The basis of this reduction is being investigated using defined mutants in type three secretion genes. The longer-term objective is to use peptide elution and mass spectrometry to examine the presentation of effector protein peptides and determine whether E. coli O157 has evolved to interfere with this process.

The yeast Candida albicans has the ability to induce several systematic and superficial diseases in the immunocompromised or immunosuppressed host. Statins are widely used drugs for the control of cholesterol biosynthesis in the body and are therefore used in treatment of hypercholesterolemia. There is increasing evidence for the potential use of statins in preventing and treating fungal infections. The aim of this study was to assess the effect of statins on C. albicans and to characterize the proteomic alterations occurring in C. albicans in response to statin. Pre-growth of C. albicans in the presence of statin lead to lower levels of ergosterol, reduced adherence to buccal epithelial cells and decreased virulence in Galleria mellonella larvae. Cells also showed increased permeability as measured by elevated amino acid and protein leakage. Proteomic analysis of C. albicans exposed to statin revealed differential abundance of proteins related to ergosterol biosynthesis such as squalene monooxygenase (4.52 fold increase), and lanosterol synthase (2.84 fold increase). Proteins involved in oxidative stress response such as small heat shock protein 21 (6.33 fold decrease) and glutathione peroxidase (2.05 fold decrease) and adherence related proteins such as yeast-form wall protein 1 (6.26 fold decrease) and Ecm33p protein (2.06 fold decrease) were also altered in abundance. These results indicate that statins have the ability to reduce the growth of C. albicans and induce an oxidative stress response. Statins may have potential to control fungal infections in vivo if used alone or in combination with conventional antifungal agents.

The pulmonary mucus of Cystic Fibrosis (CF) patients displays elevated levels of the Cathelicidin antimicrobial peptide LL-37. The interactions between the pulmonary antimicrobial peptide arsenal and Aspergillus fumigatus, a common pathogen of CF patients, have been neglected in the literature. Exposure of A. fumigatus to LL-37 and its derived fragment RK-31 (1.95 µg ml−1) for 24 h, has a stimulatory effect on growth (199.94±6.17 %, P<0.05) and (218.20±4.63 %, P<0.05) respectively, whereas scrambled (SCR)-LL-37 did not. Exposure of mycelium to 5 µg ml−1 LL-37 for 48 h increased hyphal wet weight (4.37±0.23 g, P<0.001) compared to the SCR-LL-37 treatments. The levels of Immunosuppressive metabolite gliotoxin was significantly increased at 24 h from LL-37 exposed hyphae (169.1±6.36 ng mg−1 hyphae, P<0.05) compared to the SCR-LL-37 treatments. The whole cell proteomic response A. fumigatus to LL-37 revealed an increase in proteins associated with growth (eIF-5A), tissue degradation (aspartic endopeptidase) and allergic reactions (Asp F13). By 48 h there was an increase in proteins indicative of cellular stress, growth and virulence. A. fumigatus conidia incubated in LL-37 and RK-31 displayed increased pathogenicity and fungal burden in the Galleria mellonella larvae model of aspergillosis. These results find that endogenous LL-37 paradoxically stimulates A. fumigatus growth and this may result in increased fungal growth and secretion of toxins in the lungs of CF patients.

Citrobacter rodentium murine infection is the ‘gold standard’ model for investigating host response to human diarrhoeal pathogens, Enteropathogenic and Enterohaemorrhagic Escherichia coli. Mouse strains have varying susceptibility to C. rodentium with some developing self-resolving mild diarrhoea whereas others develop severe diarrhoea and dehydration resulting in lethal disease. One of the primary defences against these invading pathogens are intestinal epithelial cells (IECs) which line the gastrointestinal tract forming a protective barrier. Previously, we have characterised changes in the metabolic landscape of C. rodentium infected IECs from resistant C57Bl/6 mice, which develop self-limiting colitis upon infection. Here, we examine the global proteomic response of infected IECs from a lethally susceptible host, C3H/HeNCrl. We highlight conserved infection signatures between resistant and susceptible mice, including the upregulation of cell cycle and DNA replication processes at the peak of infection. Temporal analysis of the IEC landscape revealed reduced expression of the differentiated Reg4+and Slc26a3 containing cells three days post inoculation (DPI), earlier than reported for C57Bl/6. Further investigation revealed the onset of other infection-induced host responses also occurring by three DPI. Bioluminescent imaging showed C. rodentium to colonise a larger proportion of the colon and microbiome analysis revealed the absence of the C. rodentium competing phyla, Bacteroides from the host-pathogen interface. Our results suggest that the absence Bacteroides from the uninfected C3H microbiome may enable C. rodentium to extensively colonise the colon accelerating the onset of host protective responses which can ultimately be of detriment and lead to increased disease severity in C3H/HeNCrl.

An investigation of Pseudomonas aeruginosa attachment to a diverse range of polymers in a high-throughput microarray format resulted in the discovery of novel biofilm-resistant and biofilm-stimulating materials. These findings raised questions about the nature of the surface interactions involved and in particular the sensory mechanisms use by P. aeruginosa to distinguish between different surface chemistries. To further investigate this, Tn5 mutants of the P. aeruginosa PAO1 Washington sub-line were tested for biofilm formation on the polymer microarrays. This revealed that a Tn5::cpxR mutant had a significant difference in biofilm formation when compared with PAO1-W. In Escherichia coli cpxR forms an operon with cpxA and cpxP. Together they form the CPX two-component signalling system controlling biofilm formation in response to cell envelope stress. To further characterise the P. aeruginosa cpxsystem, ΔcpxR,ΔcpxP and ΔcpxA deletion mutants were constructed. No significant differences were observed in their growth or in the swimming, swarming or twitch motility phenotypes normally required for surface colonization. Unlike E. coli the PAO1-W ΔcpxAmutant was able to invade lung epithelial cells and did not display increased sensitivity to antibiotics. However, the ΔcpxRmutant showed increased biofilm formation on glass and eDNA secretion in both biofilm and liquid modes of growth. This work highlights the relationship between biofilm formation and the CPX system in P. aeruginosa. However, further assays need to be conducted in order to understand the sensory mechanism(s) involved in surface sensing via the CPX system.

Haemolytic uraemic syndrome (HUS) is a complication which may arise upon enterohaemorrhagic E. coli (EHEC) colonisation of the colon. The development of HUS is associated to Shiga toxins (Stxs) release leading to organ damage. It has been found that Stx release can occur within outer membrane vesicles (OMVs). Hence, the effect of different intestinal environment cues on EHEC OMV production was examined. OMVs were purified following EHEC growth in Luria broth (LB), simulated colonic environmental medium (SCEM) with or without bile salts, and in the presence or absence of human cell lines. Yield was quantified through SDS-PAGE and densitometric analysis of outer membrane proteins F/C and A. Furthermore, OMV uptake by HEp-2 and T84 cells was analysed by incubating such cells with OMVs labelled fluorescent lipophilic dye and fluorescence microscopy. Different OMV yields were attained under different conditions, with human cell growth medium and SCEM producing significantly more than cultures grown in LB, with further increases in the presence of bile salts. The presence of HEp-2 cells did not affect OMV yield, yet a lower yield was attained in the presence of T84 cells. These results suggest that colonic environmental factors may influence EHEC OMV production in vivo. OMVs were also shown to be up-taken by both cell types. Such observations with T84 cells suggest that the human colonic epithelium can uptake OMVs. Coupled with Stx, this data may offer a paradigm on how OMVs contribute to Stx translocation across the gut barrier, subsequently leading to HUS.

Science progresses through critical evaluation of underlying evidence and independent replication of results. However, most research findings are disseminated without access to supporting raw data, and findings are not routinely replicated. Furthermore, undisclosed flexibility in data analysis, such as incomplete reporting, unclear exclusion criteria, and optional stopping rules allow for presenting exploratory research findings using the tools of confirmatory hypothesis testing. These questionable research practices make results more publishable, though it comes at the expense of their credibility and future replicability. The Center for Open Science builds tools and encourages practices that incentivizes work that is not only good for the scientist, but also good for science. These include open source platforms to organize research, archive results, preregister analyses, and disseminate findings. This poster presents an overview of those practices and gives practical advice for researchers who want to increase the rigor of their practices.

The synthesis of metal and semiconductor nanoparticles is an expanding research area due to the potential applications for the development of novel technologies. Generally, nanoparticles are prepared by a variety of chemical methods which are not environmentally friendly. This study revealed the convenient and extracellular method for the synthesis of silver nanoparticles by reducing silver nitrate with the help of onion (Allium cepa) extract. In this study preparation of silver nanoparticles by using plants extract of onion (Allium cepa) has been investigated. Characterization of different properties of the prepared nanoparticles by techniques such as, UV spectroscopy and FTIR are carried out. The antimicrobial activity of the prepared silver nanoparticles on different microorganisms such as Staphylococcus aureus and Escherichia coli was carried out. The silver nanoparticles showed antimicrobial activity against Gram positive and Gram negative bacteria. From the experiment it was found that the synthesized nanoparticles have a significant antimicrobial activity.

This study aimed to identify the bioactive compound (s) possessing both the antimicrobial and antioxidant activity. The antibacterial activity of hexane, ethyl acetate, methanol polar fraction and aqueous extract of stem of Sphaeranthus indicus was evaluated against MTCC bacterial strains Bacillus cereus-430, B. subtilis-441, Staphylococcus aureus-96, S. epidermidis-435, Escherichia coli-1687, Klebsiella pneumoniae-3384, Pseudomonas aeruginosa-741 and Proteus vulgaris-744 with their corresponding clinical isolates. The results revealed inhibitory activity of hexane extract against most of the bacterial pathogens except MTCC K. pneumonia and clinical B. cereus. The ethyl acetate extract inhibited growth of MTCC B. cereus, B. subtilis, S. epidermidis, P. aeruginosa and clinical isolates of B. cereus, S. aureus, S. epidermidis, E. coli, K. pneumonia. The methanol polar fraction exhibited activity against clinically isolated S. aureus, S. epidermidis, P. aureginosa, P. vulgaris while aqueous extracts had no activity against any of the organisms. Among all the extracts showing antioxidant activity, aqueous extract was found to possess highest activity when tested by reducing power, DMPD and DPPH assay. Phytochemical analysis revealed that methanol polar fraction had highest quantity of terpenoids while aqueous extract was rich in phenols and flavonoids. The active compound from hexane extract was isolated by TLC and the active band was detected via bioautography. DPPH was used for detecting antioxidant (s) in TLC plate. A total of 13 bands were obtained after separation in which two bands showed both the antibacterial as well as antioxidant activities. Hence it is speculated that bioactive compound showing antibacterial activity may also possesses antioxidant activity.

Antimicrobial resistant infections are at a crisis point, posing a massive threat to human health with an anticipated annual mortality of 10 million people by 2050. Antimicrobial peptides (AMPs) are a promising solution to treat drug resistant infections, with the highly competitive ecosystem of the rumen microbiome being a fruitful resource for mining AMPs. In this study, we aimed to improve the stability of a known rumen AMP with great therapeutic potential, Lynronne-1. This AMP is efficacious against topical skin infections of Methicillin resistant Staphylococcus aureus but has no activity in systemic infections when administered intravenously due to degradation by peptidases. To overcome this hurdle and improve its use intravenously, we substituted the L isoforms N and C terminal amino acid residues to d-isoforms, thereby increasing the stability of the peptide in the presence of trypsin by three-fold. The activity of the modified peptide, named Lynronne-1D against S. aureus was subsequently investigated. Lynronne-1D retained its antimicrobial activity with an MIC of 8 µg ml−1 against S. aureus and improved MICs (>4 fold) in Gram-negative bacteria strains. The peptide had rapid and potent bactericidal activity causing a ≥6 log c.f.u./ml reduction in viable S. aureus cells within 30 min of treatment. It induced membrane permeabilization within 5 min and successfully prevented biofilm formation by S. aureus cells. Lynronne-1D was also non-cytotoxic to mammalian blood cells. The improved properties of Lynronne-1D over the original peptide makes it a promising therapeutic agent for the treatment of systemic infections of S. aureus.

Antimicrobial resistance is one of the biggest global threats to human health in the modern day. No new classes of antibiotics have been approved for clinical use in over 20 years, and so called resistant ‘superbugs’ – such as Pseudomonas aeruginosa are becoming more resistant to even last resort antibiotics. Streptomyces spp. are the most valuable source of antibiotics to date, and genetic analyses has suggested that each strain is capable of producing upwards of thirty secondary metabolites. However, these genes are not all expressed in laboratory monoculture. In this study, four strains of Streptomyces were co-cultured in pairs on five different agar media. It was found that out of 108 conditions, 17 were capable of producing antimicrobial compounds that were bioactive against the ATCC ESKAPE pathogens that neither strain was capable of producing alone. Interestingly, instances of loss of phenotype were also observed, where isolates capable of bioactivity had this activity reversed when in co-culture with another streptomycete. A wild isolate of Streptomyces lydicus was also the subject of genomic and morphological characterisation in this study. Next-Generation Sequencing (NGS) of this strain was carried out using Ion Torrent, and after assembly was composed of 380 scaffolds. AntiSMASH analysis of this strain predicted 27 secondary metabolite gene clusters within the genome. Furthermore, CARD predicted 23 genes associated with antimicrobial resistance. Also presented here is a novel approach to liquid co-culture, in which modified glassware allows for the chemical interactions of two strains across a membrane, while the bacteria themselves are segregated.

Antibiotic and antimicrobial resistance is a global issue, not only for medical and veterinary treatment, but also for economic development. One of the critical bacterial pathogens known to develop resistance to antimicrobial agents is Acinetobacter baumannii, one of the ESKAPE pathogens. A. baumannii is an aerobic Gram-negative coccobacillus bacterium associated with bacteraemia, urinary tract infections and ventilator-associated pneumonia, and is typically viewed as an opportunistic pathogen. Previous research has shown that clinical strains of A. baumannii have susceptibility to novel antimicrobial peptides, specifically those identified from a rumen metagenomic dataset. Using standardised 96 well MIC testing, 3 antimicrobial peptides (Lynronne-1, Lynronne-2, Lynronne-3, identified as part of a previous research project by the Huws Lab) were compared against multiple strains of A. baumannii, in order to show efficacy of treatment against a wider variety of strains. Of the strains tested, a number were clinical isolates with demonstrated resistance (imipenem resistant, OXO-23/OXO-50). The results show that the antimicrobial peptides had noticeable inhibitory effects on the bacterial growth. There was also variation between the 3 peptides utilised, with Lynronne-1 appearing to have the lowest MIC over the majority of the strains tested. Further research as part of this project will utilise other identified antimicrobial peptides, including rationally designed peptides, as well as potentially using another gastrointestinal microbiome metagenomic dataset to identify a greater number of novel antimicrobial peptides.

Bacillus spp. produces lipopeptides that are of pharmaceutical and agricultural interest and include iturins, surfactins and fengycins. Their antimicrobial mechanism has been well studied but the role of the lipid chain and its biosynthesis is relatively unknown, especially in fengycin. In this project, we aim to fully understand the mechanisms of fengycin’s antimicrobial activity and generate new fengycins with fluorine in the lipid chain. We have already produced fluorinated lipopeptides via precursor-directed biosynthesis using fluorinated amino acids. The Bacillus genome was sequenced by MicrobesNG and a total of 34 gene clusters were identified, of which 19 were fully characterised using anti SMASH. Genes within the fengycin cluster were cleaved during the sequencing process and iturin biosynthetic genes were also present. Four fatty acyl-CoA ligases within the genome were identified using Artemis in an aim to heterologously express them, and carry out activity assays with fluorinated lipid tails. Lipopeptide production was optimised by altering culture conditions (pH, aeration and temperature) and it was found that 37 °C is the optimum temperature for biosynthesis.

Glycopeptide antibiotics are clinically important as a second-line therapy for the treatment for nosocomial infections caused by Gram-positive pathogens. A universal mode of action for glycopeptide antibiotics is to target the terminal residues, d-Alanyl-d-Alanine on the cell wall peptidoglycan intermediate lipid II, arresting the later stages of peptidoglycan biosynthesis. This weakens the cell wall, making it susceptible to rupture. A general resistance mechanism for glycopeptide antibiotics requires the core genes, vanRSHAX, that detect the presence of a glycopeptide (VanS) and upregulate genes (VanR) which orchestrate the remodelling of d-Ala-d-Ala on lipid II to d-Ala-d-Lactate (VanHAX). Glycopeptide affinity for lipid II is reduced by 1000-fold and biological activity impaired. Our previous study has shown that altering the termini of peptidoglycan precursors by VanHAX action was not sufficient for the resistance of the glycopeptide teicoplanin in S. coelicolor, which is instead mediated mainly by VanJ. Using RNA-seq, this study is designed to understand how the adaptive response in an S. coelicolor wild type (M600) and a vanJ mutant strain differ after exposure to teicoplanin. We have also compared these data with available data on the cell wall targeting antibiotics vancomycin, bacitracin and moenoymycin. By doing so, we aim to gain insight into the molecular basis of the improved activity of teicoplanin over vancomycin as well as identify novel cellular targets of teicoplanin which can help inform the design of future glycopeptides with desirable therapeutic properties.

Antibiotic discovery has stagnated since the end of the 1960s, despite an increasing level of antimicrobial resistance in the clinic. However, recently there has been a renewed interest in searching for novel antibiotics, particularly in under-explored niches. Symbiotic relationships between bacteria and eukaryotes, for example plants and insects, can prove to be a fruitful source of novel secondary metabolites. Many ant species form mutualistic relationships with antibiotic-producing bacteria to protect their colonies against parasitic invasion. However, the antibiotic-producing potential of bacteria associated with different ant species has rarely been explored in detail. We isolated 20 bacteria from the colonies of ant species in the genera Acromrymex, Cyphomyrmex and Mycocepurus, which are all fungus-farming ants, as well as Allomerus and Tetraponera genera which predate on other insects. High quality genome sequences were generated for each bacterial strain using PacBio sequencing. AntiSMASH analysis showed that many of the strains had the potential to produce many secondary metabolites, including a modified candicidin-like compound and several other clusters with low percentage homology to known antimicrobial compounds. Under standard lab conditions less than 20 % of strains show bioactivity against bacteria Bacillus subtillis and fungi Candida albicans however their genomes suggest a much higher potential. Pleiotropic methods such as growth media variations and co-culture with other microorganisms are now being used to switch on cryptic clusters in these novel isolates. Genome mining of symbiotic strains could make a valuable contribution to antibiotic discovery since such strains are constantly having to evolve in response to their host.

With antimicrobial resistance becoming an increasingly severe issue in both the developed and developing regions of the world, new strategies need to be employed to identify and characterise novel antimicrobial activity. Pseudomonas aeruginosa is a Gram negative pathogen and a major cause of opportunistic infections in burn victims and cystic fibrosis patients. Due to a wide repertoire of antibiotic resistance mechanisms, these infections are difficult to cure and thus there is a need to develop novel antimicrobial treatments. Members of the Actinobacterial genus Micromonospora produce a broad range of bioactive secondary metabolites, a number of which possess antimicrobial properties. The goal of this research is to examine the antimicrobial properties of an actinomycete strain – identified by 16S sequencing as Micromonospora –originally isolated from the Atacama desert, Chile, with a focus on identifying and characterising anti-pseudomonad activity. Bioactivity was screened for against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii and Enterococcus faecium. AntiSMASH analysis of assembled Illumina sequencing data was used to identify putative biosynthetic gene clusters. Antimicrobial bioactivity was observed, and potential antimicrobial biosynthetic gene clusters were identified through genome mining. This work has identified antimicrobial activity from a region of underexplored ecology, and highlights the importance of sampling and examining areas which have previously been considered too hostile to support life.

Understanding the role of virulence loci within pathogenic organisms can be vital in exploring the evolution of disease. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find macrophage infectivity potentiator-like proteins (MIPs) (Clark et al., 2013). MIPs are a subset of immunophilins associated with virulence in a range of micro-organisms (Norville et al., 2011). It is unknown the role they possess in non-pathogenic strains such as Streptomyces coelicolor M145. This project will identify the role of MIPs in a non-pathogenic strain through cloning, overexpression and knock out of three genes encoding putative MIP-like proteins (SCO1638, SCO1639 and SCO2620). The phenotypes will then be characterised by growth under contrasting conditions. Antibiotic production will also be measured and compared to the wild-type M145 strain. The overexpression and mutant strains will also be tested for the ability to infect amoeba using amoeba infection assays compared to a wild-type control. The results from this study will contribute to the understanding of the role of MIPs and therefore the evolution of virulence within Streptomyces species.

There is a need for novel classes of antimicrobials to be discovered in order to tackle the growing challenges of antimicrobial resistance. Deep-sea sponges are drawing much attention due to the phylogenetically diverse and dense communities of microbes that live within their tissues. Bioprospecting these sponges offers the possibility of exploring a niche environment that could contain novel classes of antimicrobials. To assess the suitability of Pheronema carpenteri (class Hexactinellida, order Amphidiscosida) and Rhabdodictyum sp. (class Hexactinellida, order Lyssacinosida) as a source of antimicrobials, cultivation-dependent strategies were employed. We assess the culturability of sponge-associated bacterial from P. carpenteri (n=3) and Rhabdodictyum sp. (n=2) using8 treatments; 4 temperature incubation treatments (4, 15, 22–25 and 28 °C), nutritional additives (Sponge spicule extract and a low nutrient heterotrophic media additive), and finally a 24 h enrichment stage. Recovered isolates were screen recovered sponge associated-bacteria isolates for bioactivity against Escherichia coli and Micrococcus luteus. Isolates demonstrating high activity were then tested against 7 clinically relevant pathogens; Staphylococcus aurerus 6571, Streptococcus pyogenes, E. coli 1077, Salmonella enterica Serovar Typhimurium LT2, Klebsiella pneumonia 681, Mycoccoccus phlei and Candida albicans. More isolates were recovered from Rhabdodictyum sp. than P. carpenteri (P<0.005). Isolates recovered from P. carpenteri demonstrate high antibacterial activity against both Gram-positive and Gram-negative strains. 112 isolates in total were found to be bioactive against M. luteus, 55 of which were active against both M. luteus and E. coli. The highest potion of bioactive compounds derived from a 15°C treatment and from the inclusion of Sponge Spicule Extract as a nutritional additive. This research presents the first attempts of bioprospecting these two species of deep-sea sponges and thus far has shown promise in their suitability.

Anaerobic rumen fungi (phylum Neocallimastigomycota) occupy the gastrointestinal tract of several herbivorous animals, and by using their powerful hydrolytic enzymes and mechanical forces they degrade plant material in the rumen, essential for rumen efficiency. The rumen microbiome represents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including antimicrobial peptides (AMPs). AMPs are promising drug candidates, and are necessary for targeting the worldwide issue of antimicrobial resistance. Rumen fluid and faecal samples were collected from various large herbivores, and fungal cultures were grown and maintained under anaerobic conditions. After roll tube culture to isolate single-zoospore cultures, sequencing of LSU was undertaken to identify the fungi to species level. Analysis of genomic data from these cultures, alongside published data was undertaken to explore the diversity of AMPs within these fungal genomes. Using functional and computational screening, potentially novel AMPs have been discovered, with isolates showing encouraging activity against some strains of bacteria. Findings indicate that the rumen microbiome may provide alternative antimicrobials for future therapeutic application.

Antimicrobial resistance (AMR) is becoming the biggest substantial threat for public health and societal implications worldwide. Coincidentally, the development of new antibiotics has decreased and downsized by the pharmaceutical companies for the last 40 years. The past decade has witnessed an increasing effort worldwide on exploration of plant-based natural products for new antimicrobial agents. The purpose of this study is to investigate the potential antibacterial activity of traditional herbal medicinal plant against resistant pathogens of public health and economic importance such as methicillin-resistant Staphylococcus aureus (MRSA) and Acine to bacter Baumannii. The broth microdilution method was used to determine the susceptibility of resistant pathogenic strains selected from WHO priority list. A total of 57 plants were chosen based on traditional knowledge and current scientific information that has been claimed to have antimicrobial activity. Preliminary results showed 75 % of screened plants inhibited the growth MRSA (NCTC 12493) some of which exhibited similarantibacterial efficiency compared to the vancomycin (positive antibiotic control), while 21 % shows an inhibitory effect against Acinetobacter Baumannii (NCTC 12156). In addition, significant synergy was observed between some of the plant extracts and vancomycin against MRSA. Further studies are needed for the warrant of these plant candidates to be further developed into therapeutic antimicrobial agents of required efficacy and safety.

The current threat of antimicrobial resistance, the surge in antimicrobial compounds rendered obsolete and the slow emergence of new classes of antibiotics havetriggeredan urgent call for novel alternatives to treat infectious diseases. The vast microbial diversity of unexplored environments and the chemical and structural variety of specialised metabolites within it stand as one of the central points to tackle this challenge. This work focuses on the exploration of the potential for antimicrobial compounds in three new Streptomyces strains – Streptomyces sp. CG885, Streptomyces sp. CG893 and Streptomyces sp. CG926- isolated in the Natural National Park Los Nevados (Colombia) and with proved production of active metabolites against several ESKAPE pathogens. To this purpose, we constructed a 16S rRNA phylogenetic tree and studied the specialised metabolite potential of these isolates using a genome mining approach [1] to predict the presence of putative Biosynthetic Gene Clusters (BGCs). Findings from this bioinformatic prediction were linked with analysis from active extracts obtained through agar plate extraction to inform prioritization of clusters. So far, the computational analysis has predicted between 68 to 93 specialised metabolite BGCs for each strain. Interestingly, most of these compounds show less than 15 % similarity to any known compound in the database and around 69 clusters seem related to previously uncharacterised RiPPs, NRPS and PKS. Further work will focus onthe validation and study of those clusters linked to compounds with potential in clinical development.

The biosynthesis of silver nanoparticles extracellularly using baker’s yeast, Saccharomyces cerevisiae and its antibacterial activity was investigated in this study. Biosynthesised silver nanoparticles were chaterized by using UV-Visible spectroscopy, which showed a distinct observed absorption peak at 429.00 nm that is attributed to the plasmon resonance of silver nanoparticles; X-ray diffraction, which determined the average size of the silver nanoparticles to be approximately 16.07 nm; presence of oval shaped silver nanoparticles determined by scanning electron microscopy; and Fourier-transform infrared, which revealed notable peaks at 3332.2, 2903.6 and 1636.3 cm−1 corresponding to the binding of the silver nanoparticles to active biomolecules, alcohols and phenols, carboxylic acids and aromatic amines respectively. The silver nanoparticles were also found to be stable for ninety days. Antibacterial activity of the silver nanoparticles was also studied. The silver nanoparticles was significantly active (P>0.05) against the test organisms at an extract concentration of 75 µg ml−1. Concentrations less than or equal to 50 µg ml−1 were not as effective as the colony forming units at this concentration, 1.61×106 for methicillin-resistant Staphylococcus aureus and 1.45×106 for Pseudomonas aeruginosa respectively were about the same range a small the colony forming units of the controls. The silver nanoparticles inhibited methicillin-resistant S. aureusmore than they inhibited P. aeruginosa. The LD50 of the synthesized silver nanoparticles after oral administration was seen to be greater than 5000 mg kg−1 body weight and is therefore thought to be safe. This study supports the use of silver nanoparticles as therapeutic agents.

Derivatives of the secondary metabolites of Streptomyces bacteria account for over half of the antibiotics currently used in the clinic. A crucial part of overcoming antimicrobial resistance (AMR) is in the development of new antibiotics that function with a novel mechanism of action, to avoid the rapid acquisition of microbial resistance. We previously reported the isolation of the new species Streptomyces formicae, from African Tetraponera penzigiplant-ants, shown to have potent activity against several strains on the World Health Organisation’s AMR watch list including MRSA. Genetic analysis of this strain revealed one of its 45 biosynthetic gene clusters, a type 2 polyketide synthase, produced all 13 formicamycins, the natural products responsible for S. formicae’s antimicrobial activity. Several regulators of the formicamycin biosynthetic pathway have been identified, including the major repressor (ForJ). Having purified ForJ, DNA binding assays were performed based on previous cappable-RNA and ChIP sequencing experiments to confirm the interaction of ForJ with different putative promoters in the cluster. By optimising gene-reporter fusion assays in this novel strain, it has been possible to characterise some of the promoters in relation to ForJ to determine the effect of its binding upon activity of the gene cluster. Such work is essential in the progression of novel antimicrobials from the laboratory into a clinic as it must be thoroughly understood how a compound is synthesised and how its biosynthetic pathway is regulated before it can be exploited for overproduction.

The discovery of streptomycin by Selman Waksman (1943) brought into focus a new avenue of drugs from natural products, i.e., actinobacterial secondary metabolites. It forms more than 60 % of total bacterial secondary metabolites (mostly from Streptomyces). Interestingly, rare Actinobacteria can produce novel secondary metabolites with unique chemical structures. With the rise of drug resistant microbes, focus on actinobacterial research has shifted towards exploring unusual niches such as those located along land-ocean boundary where freshwater mixes with saltwater. One such ecosystem is the Sundarbans mangrove, located at the apex of Bay of Bengal. The present study aims to identify novel actinobacterial species from Sundarbans which has the ability to produce unique secondary metabolites. Mangrove sediments were collected, overall bacterial diversity and secondary metabolites producing bacterial diversity were elucidated by 16S rRNA and polyketide synthase (PKS) clone library approaches respectively. Fifteen bacterial strains were isolated from the sediment using cultured approach, among which, an isolate I2 was identified based on polyphasic taxonomy. The I2 represents a new species, Myceligenerans indicum sp. nov. This new species also possess PKS genes which indicate ability to produce secondary metabolite(s). Promising antibacterial activity of this new species was found against Escherchia coli XL10, Bacillus subtilis and Vibrio chemaguriensis Iso1 especially for fraction prepared using acetone and dichloromethane (1 : 1). Spectroscopic approaches have revealed the presence of functional groups such as amide, allene, isothiocyanate and ketenimine groups.

The study of biogeography enables an understanding of the distribution patterns of biodiversity across space and time [1]. Therefore, byusing a trait-based approach, such as antibiotic production, it is possible to assess the evolutionary, geographic and ecological variables that affect the Actinobacteria specialized metabolism [2, 3]. This is particularly important as Actinobacteria isolated from marine ecosystems have been shown to be a promising source of new drugs [4, 5]. In this study, a comparative metabolomics approach using molecular networking was applied to understand the role of biogeography on the specialized metabolism of Micrococcus spp. and Pseudonocardia spp. isolated from Arctic and Antarctic marine sediments. The LC-MS/MS analysis showed differences in the specialized metabolism of phylogenetically related strains isolated from different geographic regions. These preliminary results suggest an influence on the microbial chemical space through assessing biogeographic impact. Future work on further marine ecosystems will expand our knowledge on the relationship between the chemistry and ecology of rare Actinobacteria.

The synthesis of hydrogel scaffolds with inherent antimicrobial activity has advantages for their use in tissue engineering. An ultra-short naphthalene lysine conjugated peptide, NapFFK’K’, containing naphthalene (Nap) as a molecule of high aromaticity for gel strength, phenylalanine (F) and epsilon variant lysine (K’) has previously been shown by us to self-assemble forming hydrogels with inherent antimicrobial properties against a limited number of pathogens tested. The aim of this work was to extend the antimicrobial activity studies on NapFFK’K’ including pathogenic bacteria associated with dental infections. NapFFK’K’ was synthesised using the 9-fluorenylmethoxucarbonyl Solid Phase Peptide Synthesis. Peptide purity was analysed by mass spectrometry. Hydrogel formulation was achieved by suspending the peptide in sterile deionized water followed by addition of NaOH and HCl. Hydrogels were tested at peptide concentrations of 1 %, 1.5 % and 2 % w/v against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus, and the Gram-negative bacterium Fusobacterium nucleatum. Bacteria inoculumns were exposed on hydrogel surface for 24 h. Bacterial susceptibility assay, employing the Miles and Misra method, was used to determine antimicrobial activity of hydrogels after 24 h incubation. Our results show that peptide hydrogels exhibit antimicrobial properties against both groups of bacteria but at different peptide concentrations. The 1 % peptide hydrogels was most effective against Gram-positive bacteria whereas the 2 % peptide hydrogel was effective against Fusobacterium nucleatum. Given the efficacy of the self-assembling NapFFK’K’ peptide hydrogels against oral pathogens, they may have potential use in tissue engineering approaches for regenerative endodontic treatments.

This project aims to develop Chaplins, functional amyloid proteins from Streptomyces sp., into a novel nano-thin adhesive material for the defence industry through combining existing protein-based technology with a composite partner. Chaplin proteins were extracted from a range of wild-type strains, while a synthetic promoter system was developed to express and secrete chaplins, which are typically difficult to express and purify. Chaplin proteins were found to have potentially useful adhesive properties and we have been investigating their application in optically transparent adhesives, since adhesives currently used to bond laminated transparent materials are prone to water ingress resulting in degradation of optical clarity and delamination. These adhesives could also be lighter, thinner, and have a lower environmental impact. To determine the potential of this material as a novel synbio-adhesive for bonding glass and polycarbonate we have investigated the bonding ability and transparency of natural and engineered amyloid proteins, both alone and with partner biopolymers and bulking materials. We are now exploring ways to improve the properties of the proteins by modifying the amino acid sequences, incorporating binding domains to the composite partner and via chemical modifications. Our projects has provided proof-of-concept for the use of bio-inspired amyloid protein-based materialscompounds as both adhesives and corrosion resistant metal coatings. Supported by Defence and Security Accelerator (DASA), Defence Science and Technology Laboratory (Dstl) project grants CDE100367 and ACC101824.

The purpose of this research is to further develop existing technology using engineered functional amyloid proteins for corrosion resistance by developing a coating which provides both adhesion to substrate and corrosion resistance. Pathogenic amyloid proteins have been attributed a role in certain diseases such as Alzheimer’s Disease. However, the research undertaken here involves non-pathogenic functional bacterial amyloids, using coelicolor hydrophobic aerial proteins (chaplins or Chp) produced by Streptomyces for improving corrosion resistance. Chaplins have been proven conceptually to provide corrosion resistance properties on metals and, also provide strong adhesive properties in a multi-composite systems. The composition, application and curing of the better bonding chaplin composite will now be optimised for development of a better performing functional coating, which includes inclusion of small metabolites or natural products acting as corrosion inhibitors. This would then allow for benchmarking against existing corrosion-resistant coatings. Upon determining the most relevant formulae, samples will be analysed by using microscopic techniques such as Scanning Electron Microscopy and Atomic Force Microscopy, while corrosion will be tested using immersion tests, weathering via salt-spray and in-situ scanning vibrating electrode.

Biosurfactants (BS) are amphiphilic molecules produced as a secondary metabolite by various bacteria and yeast species and are secreted extracellularly. BS have shown to work in synergy with antibiotics and also demonstrate strong antimicrobial and anti-adhesive characteristics. This coupled with their low toxicity makes them suitable candidates as combination therapies to combat skin infections. In this study, the aim was to investigate the in-vitro antimicrobial and anti-biofilm properties of mannosylerythritol lipids (MELs) produced by Moesziomyces aphidis against Staphylococcus aureus DSM-20231, Streptococcus pyogenes ATCC-19615, Staphylococcus epidermidis DSM – 28319, Pseudomonas aeruginosa DSM-3227, Escherichia coli ATCC – 25 922 and Propionibacerium acnes DSM- 1897. MELs are most predominantly used in skin creams, thus, a rationale was developed to investigate antibiotics used to treat bacterial skin infections, namely, Polymyxin B Sulphate, Neomycin, Mupirocin and Bacitracin. Minimum inhibitory concentration (MIC) values where determined for each antibiotic and BS per bacterium using the broth dilution technique based on CLSI guidelines. BS where extracted by solvent extraction and characterised using Mass Spectrometry – High Performance Liquid Chromatography, standards were quality assured using MALDI-TOF. Flow cytometry determined percentage dead versus alive for each antibiotic, BS and combination of antibiotic and BS. Scanning Electron Microscopy determined the effect of the BS on the bacterial cell walls. This study proves that BS work synergistically with antibiotics to increase the MIC of the antibiotics resulting in a substantial decrease in antibiotic use and at lower concentration. The use of BS combination therapy has the potential to reduce resistant rates and also lengthen the time taken for resistance to develop.

Fusobacterium necrophorum is a strictly anaerobic, non-spore-forming, Gram-negative Bacillus encompassing two subspecies. Fusobacterium necrophorum subsp. necrophorum (FNN) is commonly found in animals, where it is the causative agent of foot rot and abscesses. Fusobacterium necrophorum subsp. fundiliforme (FNF) is found in humans and is the causative agent of Lemierre’s disease, a condition associated with throat infections. Little is currently known about the genomic diversity of the two subspecies. Whole-genome sequences were generated for 18 Fusobacterium necrophorum isolates recovered from recurrent and persistent sort throats in UK patients. Sixty-three whole genome sequences available for Fusobacterium spp. were downloaded from GenBank and compared with the newly sequenced isolates. All-against-all comparisons of average nucleotide identity (ANI) were done to confirm species affiliation. Strains of FNN (n=6) and FNF (n=43) shared ∼97 % ANI with each other, while strains of the same subspecies shared >98 % with one another. All the newly sequenced isolates belonged to FNF. All 49 Fusobacterium necrophorum sensu stricto genomes were subject to comparative and pangenome analyses. The strains fell into two groups, with the majority of strains clustering with the type strain of FNF. Three distinct clusters of strains were identified within the FNF group, and analyses are underway to determine their potential clinical significance. Analyses of virulence-associated leukotoxin genes are also being undertaken. It is envisaged that in-depth analyses of a large collection of Fusobacterium necrophorum genomes, particularly those of FNF, will provide novel insights into pathogenesis of these fastidious anaerobic bacteria.

The human gastrointestinal tract represents a collection of prokaryotic and eukaryotic microorganisms. It has been reported that gut microbiota plays a role in health and disease. Imbalance of gut microbial communities has been implicated in several non-communicable diseases including inflammatory gastrointestinal diseases, Type 2 diabetes, and obesity. However, a role of gut microbiota in adult Southeast Asians remains largely unexplored. Herein, we present gut microbiota data obtained from Thai volunteers living in Chiang Rai Province, Thailand. We use a combination of quantitative PCR, Next Generation Sequencing (NGS), and gas chromatography-mass spectrometry (GC-MS) to investigate microbial communities, individual microbial taxa, and metabolites of NCDs volunteers. We consider prokaryotic taxa and their relationships with body mass index/waist to hip ratio and diet. We find that specific taxa of lower taxonomic levels are associated with the lean and overweight, but not the obese phenotype. We also find that microbial prokaryotic communities differ significantly amongst the different study groups.

Chlamydiae are obligate intracellular bacteria with highly reduced genomes, reflective of their abilities to sequester nutrients from the host. The host range of this phylum far exceeds that described for the Chlamydia genus which encompasses terrestrial animals; however, much of this diversity is uncultivated. Of particular evolutionary significance are members of the earliest diverging family, Candidatus Parilichlamydiaceae, comprising only uncultivated species associated with the fish gill disease, epitheliocystis. Epitheliocystis poses a significant threat to aquaculture industries globally. Due to the lack of culture systems for Ca. Parilichlamydiaceae, little is known about their biology, which also imposes further limitations on answering epidemiological questions. Gill extracts from three Chlamydiales-positive Australian aquaculture species were subject to DNA preparation to deplete host DNA and enrich microbial DNA, prior to metagenome sequencing. We obtained three metagenome-assembled Ca. Parilichlamydiaceae genomes from three different host species, and conducted functional genomics comparisons with diverse members of the phylum. Using these genomes, we developed a novel Ca. Parilichlamydia carangidicola-specific multi locus sequence analysis (MLSA) scheme to investigate genetic diversity in this species Genomic analysis revealed highly reduced genomes (∼0.8 Mbp) that share 342 orthologs with other chlamydial families, with a notable reduction in genes for synthesis of nucleotides and amino acids. MLSA revealed a high level of genetic diversity of Ca. P. carangidicola, with 20 genotypes distributed among 29 samples from four populations within the same aquaculture facility. Culture-independent genomics has provided an unprecedented insight into chlamydial evolution, pathogenicity and epidemiology. This approach could be adapted for further genomic epidemiological investigations.

Diarrhoeal disease remains a major cause of child morbidity, growth faltering and mortality in low and middle income countries (LMICs), with Campylobacter among the most common causes. Previous work has identified the major sources in the UK and US (e.g. contaminated poultry), however little is known about the risk factors and transmission routes in LMICs, where incidence is extremely high (up to 85 % of children infected before 1 year) and suggests a different epidemiology. Risk factors such as household crowding, poor sanitation, consumption of contaminated water and cohabitation with animals, all constitute potential transmission risks but their relative importance is unknown. This is a major concern as frequent or chronic enteric (re)infection is linked to significant morbidity and growth faltering in children. Comparative genomics offers a solution for untangling complex transmission networks. Campylobacter jejuni and C. coli primarily inhabit the gut of birds and mammals and signatures of adaptation can be detected in their genomes. Therefore, by sequencing the genome of human clinical isolates and faecally contaminated environments it is possible to discern its origin. Pilot genomics studies of strains from humans, animals and food in LMICs have identified genomic variation in strains that may indicate differences in source, survival, transmission or virulence (compared to the UK). In particular we have identified globally distributed strains and lineages; country-specific strains; rapid dissemination of accessory genes in specific regions; evidence of within-household spread and strains associated with asymptomatic infection or high levels of antimicrobial resistance.

Exploring and understanding the phylogeny of the Butyrivibrio group is imperative if we are ever to fully understand the consortium of ruminal microbial enzymes that are responsible for the catalysis of multifaceted reactions, such as biohydrogenation. At present, taxonomic classification of the Butyrivibrio group is based primarily on butyrate production. This approach has become antiquated with the development of sequencing technologies and downstream bioinformatics analysis. This study investigated the taxonomic relatedness and functional capacity of the ruminal Butyrivibrio group using 72 genomes. Seventy-one Butyrivibrio group genomes were obtained via JGI (the Hungate 1000 project), and one additional bacterial strain was sequenced by ourselves. A 40 marker phylogenetic tree was constructed and visualised with the interactive Tree Of Life (iTOL), and pangenome analysis conducted using Spine/ClustAGE. Orthologous gene affiliations were identified using OrthAgogue, and glycosyl hydrolase families were identified using dbCAN then aligned with Clustal Omega. Data obtained showed that three primary clades were observed, namely the genus Pseudobutyrivibrio, B. fibrisolvens, and the remaining Butyrivibrio species. Pangenome analysis and orthologous gene affiliations revealed greater diversity within Butyrivibrio than Pseudobutyrivibrio. Butyrivibrio clades consistently showed smaller core genome sizes in comparison to Pseudobutyrivibrio, with core genome percentages as low as 4 %, indicating high levels of variance. Glycosyl hydrolase alignment shows extensive sequence dissimilarity between genes on a nucleotide and amino acid level These findings suggest that the Butyrivibrio group are highly evolved to maintain competitiveness in the rumen and emphasises the need for further research into the biochemical capacity of the Butyrivibrio group.

The issue of genome minimization for a living cell has been the subject of intense study, and raises important theoretical questions and practical opportunities. We have developed an in-silico methodology, based on genome-scale models, to minimize the number of active genes encoding enzymes and transporters in a cell’s metabolic network. In the resultant minimal metabolic networks, all the remaining active genes are essential for keeping the network working to achieve the biomass value predicted by Flux Balance Analysis. We have tested our approach on a set of genome-scale metabolic models of various eukaryotic and prokaryotic organisms, but have focussed on Saccharomyces cerevisiae. The nutrient environments employed have comprised both known and automatically generated sets of nutrients and relative maximum uptake rates. The results generate more than 1000 unique minimal networks for a single condition, demonstrating the complexity of the minimization problem. We then performed a frequency analysis on the affected genes to discover patterns or similarities among the networks and the redundancy of specific pathway-related genes. In addition to this, we also evaluated the networks using a pathway-oriented robustness analysis, to see how the networks respond to random variation in the reaction fluxes. Finally, we have done a cross-species comparison of our algorithm’s results to highlight some of the homologous genes that are retained in the networks of a majority of species. Thus, our work has produced a tool for in silico genome minimization that permits the discovery of mandatory genes in the minimal metabolic networks.