- 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.