- Volume 1, Issue 1A, 2019

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.