- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Missing Microbes and the Hygiene Hypothesis: New Challenges and Perspectives
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Identification and characterisation of the key virulence determinants of asymptomatic pathogenic Escherichia coli
More LessDiarrhoeal 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.
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- Non-human Pathogens
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Characterization of keratinophilic fungal species and other non-dermatophytes in hair and nails samples in Riyadh, Saudi Arabia
More LessThe 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.
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Stage-specific gene regulation in Perkinsus olseni parasites
More LessPerkinsus 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.
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The study of rotavirus phylogenetic diversity, re-assortment and interspecies transmission and associated virome in Northern Irish livestock and wildlife
More LessBackgroundRotavirus (RV) is a highly infectious pathogen of livestock causing diarrhoea and dehydration, and substantial economic loss. RV is an RNA virus with a segmented genome that has evolved significantly creating diverse strains. RV is believed to be endemic within livestock and the genotyping of positive isolates will allow the diversity of the rotavirus to be phylogenetically mapped for evidence of re-assortment and interspecies transmission. Rotavirus positive samples and rotavirus negative samples were examined using a metagenomics approach through next generation sequencing (NGS) to study the virome of symptomatic animals.
MethodsRV in symptomatic livestock, and a small subset of wildlife and exotic animal faeces samples (n=326), originating from Northern Ireland, were screened by RT PCR for the RV VP6 gene. NGS libraries using a de novo metagenomics approach were run on the MiSeq reagent V3 600 cycle as pairend reads. Data was quality checked by Fast QC, assembled in SPAdes, processed through NCBI blast (n) and then MEGAN to display the taxonomical content.
ResultsThe prevalence of RV VP6 gene was n=108 (33 %). Initial Sanger sequencing results showed porcine G3, G4 and G5 and bovine P7 and P13 strains. Preliminary metagenomic results indicates re-assortment and interspecies transmission with a positive bovine sample showing RV acquired re-assortment from human and equine RV strains. Analysis shows there is a large viral community present in both RV positive and RV negative animals. The metagenomic profiling through NGS data set will give a better understanding of the symptomatic virome in livestock.
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Biofilm production and other virulence factors in Streptococcus spp. isolated from clinical cases of bovine mastitis in Poland
More LessMastitis 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.
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Characterizing avian pathogenic Escherichia coli from diagnostic cases in Georgia, USA – comparison of gene profiles with tissue of isolation
More LessColibacillosis 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.
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A longitudinal survey of the gut virome of commercial broiler chickens from prehatch to slaughter
More LessUneven 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.
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Studying a mycovirus from Dothistroma septosporum, causative agent of pine needle blight
More LessDothistroma 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.
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Non-sporulating variants of C. difficile from animal faecal samples
More LessClostridioides 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.
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Brucella sequence Type 27 isolated from Dwarf Sperm Whale (Kogia sima) stranded in the Costa Rican Pacific Coast
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.
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- Offence and Defence
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The role of copXL in community acquired methicillin resistant Staphylococcus aureus USA300 hyper-resistance to antibacterial copper toxicity
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.
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Correlating prophage presence in Helicobacter pylori with restriction-modification systems
More LessThe 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.
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Bacterial lipocalins protect bacteria from antibiotic-induced membrane lipid peroxidation
More LessBurkholderia 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.
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Mechanisms underlying Enterohaemorrhagic Escherichia coli O157: H7 manipulation of the bovine cellular immune response
More LessEnterohaemorrhagic 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.
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An analysis of the role of statins in reducing the virulence of Candida albicans
More LessThe 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.
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The Cathelicidin antimicrobial peptide (LL-37) stimulates the growth and pathogenicity of the pulmonary lung pathogen Aspergillus fumigatus
More LessThe 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.
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Citrobacter rodentium triggers the rapid onset of conserved infection signatures in a susceptible mouse strain
More LessCitrobacter 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.
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Elucidating the contribution of the Pseudomonas aeruginosa CPX system in biofilm formation
More LessAn 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.
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The impact of the colonic milieu on enterohaemorrhagic E. coli outer membrane vesicle production
More LessHaemolytic 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.
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- Teaching Microbiology in Higher Education Symposium
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Practical open science: tools and techniques for improving the reproducibility and transparency of your research
More LessScience 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.
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- The Biological and Chemical Tales of the Antibiotic Makers
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Biosynthesis and antimicrobial activities of Allium cepa silver nanoparticles against some clinical isolates
More LessThe 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.
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In vitro screening of antibacterial and antioxidant properties of stem of Sphaeranthus indicus Linn
More LessThis 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.
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Investigating the efficacy and improved stability against Staphylococcus aureus of Lynronne-1D, a modified rumen microbiome derived antimicrobial peptide
More LessAntimicrobial 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.
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The emergent properties of streptomyces observed during co-culture and the genomic and morphological characterisation of a Streptomyces lydicus strain
More LessAntimicrobial 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.
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Utilising novel antimicrobial peptides identified from the rumen microbiome as a potential treatment for multiple A. baumannii strains
More LessAntibiotic 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.
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Production of fluorinated fengycins in Bacillus spp.
More LessBacillus 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.
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Understanding the adaptive response of Streptomyces coelicolor to the glycopeptide antibiotic teicoplanin
More LessGlycopeptide 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.
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Characterising the inhibition profile of an antimicrobial produced by Enterococcus
IntroductionAntibiotic resistance is one of the greatest problems facing the 21st century with few new classes of antibiotics being discovered. The forefront of antibiotic discovery has been the soil microbiome and it is still a valuable resource for identifying microbes with possible antibiotic producing capabilities leading to novel classes of antibiotics. This justifies continuing investigation into the soil microbiome for antibiotic producing bacteria, to help tackle the growing trend in antibiotic resistance. A bacterial soil isolate was found to inhibit Enterococcus faecalis ATCC 29212 and the aim of this work was to further characterise the inhibition profile of the antibacterial.
MethodsBacterial plug and supernatant assays were used to access the inhibition ability of a soil bacterial isolate against the WHO ‘priority pathogens’. Identification of the bacteria was carried out using 16S rRNA and whole genome sequencing. Synergy tests were carried out using broth dilution assays.
ResultsThe soil isolate (N5) was identified as Enterococcus and showed antibacterial activity against Staphylococcus aureus MRSA and E. faecalis VanA. This antibacterial is secreted into the supernatant which still showed inhibitory activity against MRSA and VRE. Synergy with N5 and ciprofloxacin (0.2 µg ml−1) against E. faecalis ATCC 29212 was also observed.
ConclusionBoth VRE and MRSA are important players in nosocomial infections and are displaying high levels of resistance. Further study of this antibacterial could lead to the development of a new compound to help overcome resistance mechanisms or a novel antimicrobial.
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Genome mining of ant-associated bacteria to identify novel secondary metabolites
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.
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The antimicrobial activity of Micromonospora sp.
More LessWith 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.
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Streptomyces coelicolor M145 MIP-like proteins: to identify their role in a non-pathogenic organism
More LessUnderstanding 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.
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Assessing and optimising culturing methods for the associated-bacteria of two species of deep-sea sponges (class Hexactinellida) for antimicrobial bioprospecting
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.
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Identifying novel antimicrobials from anaerobic rumen fungi
More LessAnaerobic 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.
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Antibacterial activity of traditional herbal medicine
More LessAntimicrobial 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.
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Unmasking the potential as antibiotic makers of three Streptomyces strains isolated in a high-altitude ecosystem in Colombia
More LessThe 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.
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Biosynthesis of silver nanoparticles using baker’s yeast, Saccharomyces cerevisiae and its antibacterial activities
More LessThe 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.
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Deciphering the regulatory mechanisms of formicamycin biosynthesis in Streptomyces formicae, a novel antibiotic against MRSA
More LessDerivatives 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.
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A novel actinobacterium species from a mangrove ecosystem- antibacterial activity and chemical characterization
More LessThe 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.
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Assessing metabolite biogeography of Micrococcus spp. and Pseudonocardia spp. isolated from marine environments
More LessThe 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.
References1. Lomolino, M. V., Riddle, B. R. and Whittaker, R. J. Biogeography. (Sinauer, 2016).
2. Morlon, H. et al. The biogeography of putative microbial antibiotic production. PLoS One 10, 1–15 (2015).
3. Krause, S. et al. Trait-based approaches for understanding microbial biodiversity and ecosystem functioning. Front Microbiol 5, 1–10 (2014).
4. Fenical, W. and Jensen, P. R. Developing a new resource for drug discovery: Marine actinomycete bacteria. Nat Chem Biol 2, 666–673 (2006).
5. Hug, J. et al. Concepts and Methods to Access Novel Antibiotics from Actinomycetes. Antibiotics 7, 44 (2018).
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Antimicrobial activity of naphthalene lysine conjugated peptide hydrogels
More LessThe 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.
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Development of bio-inspired protein-based materials as novel transparent adhesives for glass laminates
More LessThis 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.
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Development of advanced corrosion-resistant coatings with synthetic biology-inspired protein technologies
More LessThe 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.
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Antimicrobial and antibiofilm potential of biosurfactants as novel combination therapy against bacterium that cause skin infections
More LessBiosurfactants (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.
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- The Microbial Pangenome
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Whole-genome sequence analyses of Fusobacterium necrophorum
More LessFusobacterium 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.
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Different oral niches give rise to varying levels of genetic diversity in Streptococcus mitis
More LessBackgroundThe commensal bacterium, Streptococcus mitis is a major coloniser of the oral cavity. This close cousin of Streptococcus pneumoniae has been identified to occupy a range of oral niches independent of pH, epithelial surface and redox state. We have previously observed a high level of genetic diversity in S. mitis with an open pan-genome and only 46 % of the genome identified as core.
AimWe aim to identify the driving force behind the high levels of genetic diversity identified in S. mitis and determine if this diversity can be attributed to oral location.
MethodsSamples were collected from the buccal mucosa and tongue dorsum. A total of 75 S. mitis isolates were whole genome and Ion Torrent sequenced and subject to bioinformatic analysis.
ResultsIn contrast to S. pneumoniae, genetic diversity in S. mitis is predominantly driven by mutation with recombination playing only a minor role. It was also observed that isolates collected from the tongue dorsum were significantly more diverse than those collected from the buccal mucosa. Due to the mutational nature of S. mitis diversity, this suggests varying pressures on S. mitis in these two environments. Study is now underway to establish the genetic differences between S. mitis from these oral niches and determine how this will impact future study and study design of this significant oral microbiome component.
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Gut microbiota in Northern Thai populations
More LessThe 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.
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Genomic and epidemiological insights into chlamydial epitheliocystis
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.
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Local genes, for local bacteria: biogeographical variation in Campylobacter accessory genome content
More LessDiarrhoeal 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.
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Understanding the evolution and metabolic capabilities of the Butyrivibrio group
More LessExploring 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.
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Genome size reduction: design and analysis of a minimal metabolic network for yeast
More LessThe 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.
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