- Volume 2, Issue 7A, 2020
Volume 2, Issue 7A, 2020
- Abstracts from Annual Conference 2020
-
- Poster Presentation
-
-
The Missing Link: Developing a pipeline for accelerated antibiotic discovery from Streptomyces through linking ‘omics data
More LessThe genus Streptomyces has proven to be a rich reservoir of specialized metabolites, accounting for 80% of all microbially produced antibiotics including chloramphenicol and nystatin from S. venezuelae and S. noursei respectively. However, the discovery of novel microbial chemistry is still greatly needed to combat antimicrobial resistance. Comparative metabolomics, using platforms such as Global Natural Products Social Molecular Networking (GNPS), as well as tools such as antiSMASH and BiGSCAPE have aided the mining of biosynthetic gene clusters (BGC’s) across datasets but comparing the chemistry to the encoding biosynthetic gene clusters is a significant bottleneck.
In this study, ten Streptomyces strains were selected, based on phylogeny and availability of genome sequence. The strains were cultured on 6 types of Actinomycete-specific media to maximise metabolite diversity. Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was used to obtain spectral data from crude metabolite extracts enabling comparative metabolomics analysis via the GNPS platform. As the genome sequences were publicly available, genome mining of BGC’s was achieved using antiSMASH resulting in 260 BGC’s across the ten strains. This revealed 53 gene cluster families when analysed using BiGSCAPE, the largest encoding for 8 metabolites.
In future, both biosynthetic (BGC’s) and chemistry (parent ions) datasets will be computationally linked based on strain presence/absence. The development of standardised datasets that enable cross-‘omics comparison will aid prioritisation of novel antibiotics, especially when combined with bioactivity data.
-
-
-
The complete sequence, annotation and comparative analysis of the Escherichia coli IncFII family plasmid pMccB17, encoding biosynthetic and immunity functions for the antimicrobial peptide microcin B17
More LessMicrocin B17 (Mcb17) is a ribosomally synthesized and post-translationally modified peptide (RiPP), produced by Escherichia coli, that inhibits bacterial DNA gyrase in a similar way to quinolones. The Mcb17 operon, consisting of seven genes encoding biosynthetic and immunity/export functions, was originally found on a plasmid, pMccB17. This circular plasmid, previously known as pRYC17, was originally found in Escherichia coli strain LP17, isolated from the intestinal tract of a healthy newborn at Hospital La Paz, Spain and was transferred by conjugation to E. coli K-12 [Baquero et al. (1978) J. Bacteriol. 135: 342]. pMccB17 is a low copy number IncFII plasmid in the same incompatibility group as R100 and R1. Not much is known about this plasmid aside from the facts that it carries the Mcb17 operon, does not possess any conventional antibiotic resistance markers and its size was estimated to be approximately 70 kb.
We extracted the plasmid from E. coli K-12 strain RYC1000 [pMccB17] and sequenced it twice using an Illumina short-read method, firstly together with the host bacterial chromosome, then plasmid DNA was purified and sequenced separately. PCR primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete sequence has 83 predicted genes, initially identified by Prokka and subsequently manually reannotated using BLAST. Comparison to other IncFII plasmids shows a large proportion of shared genes, especially in the conjugative plasmid backbone. However, pMccB17 which is a MOBF12 plasmid lacks transposable elements and in addition to the Mcb17 operon, this plasmid carries 25 genes of unknown function.
-
-
-
MORF: An online tool for exploring microbial cell responses using multi-omics analysis
More LessWith continuing improvements and reducing costs of high-throughput technologies, microbiologists are increasingly collecting multi-omics datasets. However, the tools and techniques used to analyse these kinds of data are often highly specialised and require bioinformatics, statistics and often coding experience. Many studies also tend to report on a single aspect of the data whilst overlooking other potentially interesting phenomena. Consequently, many of these multi-omics data sets are not being used to their full potential. MORF was created as a solution to these problems by providing access to multi-omics datasets through an online interface which presents the data in a user-friendly and accessible way. No coding experience or specialist statistical knowledge is required, and users are free to explore the data using interactive graphics and simple analysis tools.
Here we demonstrate MORF using multi-omics datasets from two experiments using bacteria in industrial fermentation processes. First, Escherichia coli engineered to produce styrene, a valuable chemical used in the manufacture of polymers, and secondly a Clostridium which produces the biofuel butanol. A key outcome was the identification of targets believed to be involved in responding to membrane stress, which we identified using MORF’s differential gene and protein analysis tools. Work is underway to further characterise and engineer these targets to improve product yields. In conclusion, MORF provides a framework for omics analysis that can be applied to any organism or set of experimental conditions, and will help researchers and collaborators to make the most of their data.
-
-
-
Modulation of cAMP levels by a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria
More LessAntimicrobial tolerance is the gateway to the development of antimicrobial resistance and is therefore a major issue that needs to be tackled.
The second messenger, cyclic-AMP (cAMP) is conserved across all taxa of life. It is involved in propagating the signal from environmental stimuli and converting it into a response. In bacteria such as M. tuberculosis (Mtb), P. aeruginosa, V. cholerae and B. pertussis, cAMP has been implicated in virulence, regulation of metabolism and gene expression. Cyclic AMP signalling in mycobacteria is especially complex – with 16 enzymes that produce cAMP in Mtb alone.
By discovery of a novel, actinobacteria conserved enzyme that degrades cAMP, we have developed a tool to modulate cAMP levels in mycobacteria. By using a combination of metabolomics, bioenergetics and time-to-kill assays, we show that when this enzyme is overexpressed in the model organism M. smegmatis, there is a 3.3 -fold decrease in intracellular cAMP levels. This was concomitant with 7-fold increased ATP. The unbalanced ATP/cAMP ratio consequently altered cell envelope permeability, compromised bioenergetics and most importantly, led to a decrease in the tolerance to various frontline antimicrobials.
Taken together, this work provides clear evidence that cAMP is involved in antimicrobial tolerance in mycobacteria and that this may represent a promising new target for antimicrobial development.
-
-
-
Studying the surface-tethered AaaA as a virulence factor and anti-P. aeruginosa drug target
More LessP. aeruginosa is a leading cause of bacterial wound infections, and is associated with a disproportionally high level of mortality in burn patients especially. Because of its wide arsenal of virulence factors and biofilm formation ability, infections with P. aeruginosa are often chronic and extremely difficult to treat. This study uses an ex-vivo skin model to study the role of the virulence factor AaaA (PA0328) in chronic wound infections. AaaA, or the Arginine-specific aminopeptidase of Pseudomonas aeruginosa A, is a surface-tethered autotransporter which cleaves N-terminal arginine from peptides. In the oxygen and nutrient-limited environments of chronic wounds, this free arginine could serve as a nutrient source for P. aeruginosa via alternative metabolic pathways. Changes in local arginine concentrations may also alter host iNOS and Arginase immune responses which influence inflammation and wound healing, in order to favour a chronic infection. Being surface-tethered and immunogenic, AaaA is also of interest as a potential antimicrobial drug or vaccine target. This study aims to probe the role of AaaA on both pathogen survival and host response in the wound context, using a combination of in situ transcriptional reporters, immunofluorescence and laser-scanning microscopy, and RT-qPCR to localise and quantify P. aeruginosa survival and aaaA expression, as well as resident immune cell invasion, and expression of immune factor genes, in wounds over time. Here, we present preliminary data examining AaaA expression and function in P. aeruginosa biofilm cultures and ex-vivo skin wound infections, as well as the results of preliminary inhibitor screens.
-
-
-
Structural characterization of genomic RNA-coat protein contacts in single-stranded RNA viruses by high-resolution cryo-EM
Recent developments in cryo-electron microscopy (cryo-EM) hardware along with continuously evolving software tools have led to the discovery of many novel structures that it was not possible to solve until now, resulting in what is termed “the resolution revolution”. In structural virology, it has also led to a re-evaluation of known structures. Most virion structures solved by X-ray crystallography or cryo-EM are focused on the capsid protein (CP) as a result of the application of icosahedral symmetry averaging to “improve” the electron density maps. However, this has the consequence that the intrinsic asymmetry of important components of virions, such as the viral genome and structural proteins lacking such symmetry, are masked. Single-stranded (ss), positive-sense RNA viruses are major pathogens in all kingdoms of life. Asymmetric cryo-EM structure determination of a major model virus in this class, bacteriophage MS2, reveals the limitations of a symmetrized view. As well as the presence and interactions made by the unique Maturation Protein, it also reveals multiple gRNA-CP dimer contacts corresponding to our previous prediction that dispersed, sequence-degenerate RNA motifs (Packaging Signals, PSs) play important roles during the virion assembly. Here, we describe how relaxing symmetry during structure determination can image such gRNA-PS contacts in a range of ssRNA viruses including the picornavirus Bovine Enterovirus-1, the alphaviruses Sindbis and Semliki Forest Viruses, as well as the plant virus Turnip Crinkle Virus. The revelation of these functionally important gRNA-CP contacts changes our fundamental understanding of assembly in these pathogens and may have further translational importance.
-
-
-
An image-based screening system for the study of Mycobacterium avium persistence and drug discovery
More LessMycobacterium avium infects human macrophages causing opportunistic infections. A steady increase of these infections over the past four decades and resistance to common anti-mycobacterial drugs, create an urgent need for new treatments; however, drug discovery is held back by a lack of knowledge about how M. avium replicates or persists in host cells.
We implemented an image-based assay using a fluorescence dilution (FD) system to measure M. avium replication and persistence. M. avium strain 104 carrying a plasmid encoding GFP and TurboFP635 under constitutive and inducible promoters, respectively, is induced prior to infection of THP1 macrophages and the fluorescent signals are tracked over time in the absence of the inducer. Loss of the TurboFP635 signal while GFP signal is maintained, identifies replicating and retention of both signals non-replicating bacteria.
In the absence of inducer, the M. avium 104 FD strain replicated in the macrophages, leading to increasing numbers of GFP-expressing intracellular bacteria and concomitant loss of TurboFP635 signal in >90% of the infected cells after 24 hours. Upon re-induction, these bacteria expressed TurboFP635, suggesting they are metabolically active and alive. We observed the presence of a small non replicating population that persisted over 96 hours pi. We applied our assay to compare the effect of a panel of anti-mycobacterial drugs, revealing different effects on killing, intracellular replication and induction of persisting, non-replicating bacteria, illustrating the power of this system to facilitate the dissection of the biology of persistence and anti-mycobacterial drug discovery in the future.
-
-
-
Taking charge: Using electrochemical impedance spectroscopy to quantify biofilm formation in Pseudomonas spp.
The opportunistic pathogen, Pseudomonas aeruginosa, is infamous for its ability to rapidly form biofilms (<24 h) in inhospitable environments and the development of antimicrobial resistance (AMR). It has been seen to have resistance to nearly all known antibiotics, including last-line antibiotics colistin and carbapenems. AMR is currently considered one of the biggest threats to human health, causing 700,000 deaths annually, expected to rise to 10 million deaths per year by 2050. P. aeruginosa, alongside other opportunistic pathogens, has been implicated in infections following various surgical procedures. Such infections compromise patient recovery and, when a medical implant is present a biofilm can develop that will ultimately require a complex revision surgery to remove the infection.
In this study, impedance spectroscopy and differential pulse voltammetry were carried out in parallel to measure electrochemical and impedance properties of bacteria, allowing for identification and quantification of pyoverdine and pyocyanin; bacterial metabolites. Three Pseudomonas spp. (P. aeruginosa, P. fluorescens and P. putida) were assayed in liquid culture at OD600. The sensor was standardised with pyoverdine and pyocyanin, with an electrochemical reading taken every 30 minutes up to 4 hours. This assay was repeated with Pseudomonas spp. growing in biofilms in LB broth, with a screen-printed electrode as the solid surface. Readings were then used to correlate metabolite production to biofilm production in each Pseudomonas sp. Pyoverdine correlated with biofilm formation for all three assayed Pseudomonas, with variation in the quantity of metabolite produced between species. This allows the two metabolites to be used as indicators of biofilm mass on devices and surfaces.
-
-
-
Incidence of malaria parasites in human immuno-deficiency virus clients attending General Hospital Awo-Omamma, Imo State Nigeria
More LessThe incidence of malaria parasite in Human Immuno-deficiency Virus clients attending Awo-omamma General Hospital, Owerri Imo state Nigeria was studied. A total of 200 blood samples were collected; 150 samples were collected from sero-positive HIV clients while 50 samples were collected from sero-negative HIV clients which served as control samples. Out of this 200 clients 85(42.5%) were males while 65(32.5%) were females. The blood samples were analyzed using Malaria Rapid Test Kit for the presence of Plasmodium falciparum, using standard medical laboratory procedure. The result revealed an overall prevalence of 43 (28.7%) for HIV positive participants that tested positive to malaria parasite, 15 (17.6%) were male while 28 (43.1%) were female. Analysis based on age revealed that the highest prevalence was among those within the age group 30-39 years having 20 (10%) while those with the least prevalence were observed among those within the age group 20-29 years having 32 (16%). Analysis of malaria parasite based on CD4+ cell count among HIV clients revealed that 51(34%) had CD4+ cell count above 200cell/μl while 23 (15.3%) had CD4+cell count below 200cell\μl. This study has shown that there is a low prevalence of malaria parasite (Plasmodium falciparum) among HIV/AIDs clients with high CD4+ attending HIV clinic in Awo-omamma General Hospital, Imo state. It is recommended that more efforts be made to eradicate malaria completely as this will go a long way in reducing the rate of mortality among HIV clients.
-
-
-
Increasing production of the antibiotic-abyssomicin C in Micromonospora maris
More LessAbyssomicin C is a polyketide antibiotic produced by Micromonospora maris AB-18-032. Previous work on abyssomicin C indicated that it inhibits growth of infectious pathogens such as Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). It does this by suppressing para-aminobenzoic acid (pABA) synthesis, which is required for folic acid biosynthesis in bacteria. This makes abyssomicin C an appealing antibiotic drug, as it is specific only to bacteria. However, its yield in chemical synthesis (4 %) and biosynthesis (60.0 mg/L) are low. Ribosome engineering, through the selection of streptomycin-resistant and rifampin-resistant mutants, of M. maris may result in strains with a higher titer of production. After screening by bioassay and sequencing, the mutant genes in six Ochi mutants were identified. Of these mutants, four of them are able to produce a higher titer of abyssomicin C. The other two strains were detected to produce the other secondary metabolite.
-
-
-
Ultra- structure of Toombak; smokeless tobacco of Sudan and its effects on oral and systemic health
More LessIntroduction: Toombak is a smokeless tobacco used by the Sudanese. Nicotiana Rustica leaves are fermented, and sodium bicarbonate is added, increasing Ph. This causes high absorption spikes of unprotonated nicotine through mucosal epithelium, and into the blood, distinguishing Toombak as an addictive product. Furthermore,Toombak hashighmicrobial contamination due to its open field, rural production.
Materials and methods: 21 tobacco samples were collected from 3 main towns in the capital Khartoum, Sudan. Analysis included microbiome (16S rRNA sequencing), metabolome (Liquid and Gas Chromotography- volatile organic compound method), heavy metal content and scanning electron microscopy (SEM).
Results: Compared to limit of detection values (LOD), Tobacco specific nitrosamines (TSNA’s), in particular, 4(methylnitrosamino)-1-(3-pyridyl)–butanone (NNK) were markedly increased [NNK; 1.2-4.7 mg/g; LOD 0.02]. Free nicotine ranged from 16-31 mg/g; [LOD 0.01]. High choline and carnitine, volatile aldehydes; and benzyl alcohol were also detected. 8 cations, of which iron [1465 mg/kg] and copper [3.76 mg/kg] were strikingly observed, compared to average levels from other products; [Iron 20-60mg/kg, Copper 1.3mg/kg]. 8 Phyla, including but not limited to; Actinobacteria; cornyebacterium(contain nitrate reductase genes) and Firmicutes; staphylococcus and facklamia, were sequenced. SEM highlighted a non-homogenous product with elevated sodium spectrum.
Conclusions: TSNA’s were potent in Toombak due to high starting nicotine and rich nitrate reductase bacteria. High choline and carnitine can promote cardiovascular disease through their conversion to trimethylamine-N-oxide. Diphtheria and infective endocarditis are associated with cornyebacterium and facklamia respectively while staphylococcus can lead to numerous systemic opportunistic diseases.
-
-
-
AMR Escherichia coli and its temporal and spatial variability within the aquatic environment
More LessPhenotypic and genotypic identification methods have been used to determine the temporal and spatial dynamics of AMR-Escherichia coli in a mainly rural watercourse that receives WWTP-effluent compared to a parallel river which does not. We aimed to investigate the incidence of plasmid-mediated mcr-1and β-lactamase-genes in E. coli recovered from both water and Asellus aquaticus samples throughout two-calendar-years.
Samples of the water and A. aquaticus were recovered from the relevant locations each month. CHROMagar ESBL agar was used throughout to isolate and identify ESBL-E. coli. The presence of AMR-genes was confirmed using the ‘BSAC’ antibiotic-disk-synergy method and PCR analysis to confirm the presence of mcr-1and ESBL-genes. The CHROMagar ESBL agar was found to be 99.7% (n=578) accurate when confirmed with a PCR analysis of the ESBL-genes. Seventy-six-point-six percent (n=449) of the isolated ESBL-E. coli were correctly identified as ESBL-producing organisms using the ‘BSAC’ method. Interestingly 61.9% (n=358) of the ESBL-E. coli were also found to carry the mcr-1 gene.
Our data shows that AMR levels were highest at the WWTP-effluent throughout the two-years for both water and A. aquaticus samples. The incidence of AMR-E. coli 1km downstream of the effluent discharge was equivalent to the parallel river sites, suggesting that the dispersal of AMR from the WWTP-effluent is limited, although AMR-E. coli were found in relatively high numbers at the WWTP-effluent. We argue that the presence of AMR in the freshwater invertebrate A. aquaticus could represent an important route by which AMR can spread from the aquatic environment to the terrestrial environment.
-
-
-
Identification of genes that contribute to fitness of African and Global clades of Salmonella Enteritidis during infection of macrophages
More LessNon-typhoidal Salmonella (NTS) usually cause gastroenteritis in humans, but in recent years NTS have begun to cause epidemics of bloodstream infections in Africa. Salmonella Enteritidis is the second most common serovar associated with this invasive form of NTS disease (iNTS) in Africa. To establish a systemic infection, Salmonella must survive and replicate within host cells, with macrophages being a primary target. Genomic characterisation of S. Enteritidis isolates from human bloodstream has identified two new clades that are unique to Africa and distinct from the Global Epidemic clade. The African S. Enteritidis clades exhibit genomic degradation, and possess a distinct prophage repertoire and are multi-drug resistant. However, little is known about the virulence factors that allow African S. Enteritidis to cause systemic infection in susceptible hosts. We screened libraries of random insertion mutants of African and Global S. Enteritidis by transposon insertion sequencing (TIS), and identified about 280 genes belonging to each clade that contribute to bacterial survival and replication in murine macrophages. The genes were associated with 5 pathogenicity-islands, or encoded the global regulators PhoPQ and OmpR-EnvZ. Experiments are ongoing to investigate the role in intra-macrophage replication of genes that are uniquely identified in African Salmonella. It is hoped that our findings will contribute to a greater understanding of African Salmonella infection biology, and that some of the virulence-associated genes could be potential targets for novel therapeutics.
-
-
-
Bifidobacterium adolescentis shows potential to strengthen host defence against gastrointestinal infection via inhibition of the opportunistic pathogen Candida albicans and stimulation of human-isolated macrophages killing capacity in vitro
The human gut microbiota enhances the host’s resistance to enteric pathogens via colonisation resistance, a phenomenon that is driven by multiple mechanisms, such as production of antimicrobial metabolites and activation of host immune responses. However, there is limited information on how individual gut bacterial species, particularly many of the dominant anaerobes, might impact the host’s defence.
This study investigated the potential of specific human gut isolates to bolster the host’s resistance to infection. First, by antagonising the opportunistic fungal pathogen Candida albicans, and secondly, by modulating the killing capacity of human-isolated macrophages in vitro.
Co-culturing C. albicans with faecal microbiota from different healthy individuals revealed varying levels of fungal inhibition. In vitro assays with a panel of representative human gut anaerobes confirmed that culture supernatants from certain bacterial isolates, in particular of Bifidobacterium adolescentis, significantly inhibited C. albicans growth. Mechanistic studies revealed that microbial fermentation acids including acetate and lactate, in combination with the associated decrease in pH, were strong drivers of this inhibitory activity.
In the second in vitro assay, human-isolated macrophages were exposed to bacterial supernatants, and subsequently tested for their capacity to eliminate adherent-invasive Escherichia coli. Among the gut anaerobes tested, B. adolescentis was revealed to exert the strongest immunostimulatory and killing effect when compared to the unstimulated macrophages control.
B. adolescentis is known to be stimulated by dietary consumption of resistant starch andmay therefore represent an attractive target for the development of probiotic and prebiotic interventions tailored to enhancethe host’s natural defences against infection.
-
-
-
A horizontally gene transferred copper resistance locus enables survival of community acquired methicillin resistant Staphylococcus aureus USA300 in host cells
The spread of community acquired, methicillin resistant Staphylococcus aureus (CA-MRSA) is an increasing problem seen outside the healthcare setting. One such strain, CA-MRSA USA300, is epidemic in the United States. USA300 shows a heightened resistance to the innate immune system, in particular to macrophage engulfment. Two horizontally acquired genes, encoding an efflux pump (CopX) and lipoprotein (CopL), were discovered in 2 different lineages of USA300, representing CA-MRSA epidemics in North and South America. Removal of either of these genes resulted in elevated copper concentrations in the cytoplasm of S. aureus, implying a function in copper hyper-resistance. While copper is an essential part of metabolic machinery, it is toxic at high concentrations and is utilised by macrophages to kill bacteria in the phagosome. Supporting this, USA300 with functional copXL genes showed increased survival in macrophages compared to their copXL negative counterparts. Although the role of CopX as an efflux pump explains the rise in intracellular copper concentration upon its mutation, the role of the CopL lipoprotein is still unknown. Therefore, to better understand the function of CopL and how it might influence S. aureus host interaction, transcriptomic analysis is underway to identify downstream targets. This has the potential to uncover an exciting mechanism linking metal resistance to host virulence.
-
-
-
Non-human primates in the Gambia harbour human-associated pathogenic Escherichia coli strains
Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between different host species. We have investigated genetic diversity and antimicrobial resistance in Escherichia coli isolates from a range of non-human primates dispersed across the Gambia: patas monkey (n=1), western colobus monkey (n=6), green monkey (n=14) and guinea baboon (n=22). From 43 stools, we recovered 99 isolates. We performed Illumina whole-genome shotgun sequencing on all isolates and nanopore long-read sequencing on isolates with antimicrobial resistance genes. We inferred the evolution of E. coli in this population using the EnteroBase software environment. We identified 43 sequence types (ten of them novel), spanning five of the eight known phylogroups of E. coli. Many of the observed sequence types and phylotypes from non-human primates have been associated with human extra-intestinal infection and carry virulence characteristics associated with disease in humans, particularly ST73, ST217 and ST681. However, we found a low prevalence of antimicrobial resistance genes in isolates from non-human primates. Hierarchical clustering showed that ST442 and ST349 from non-human primates are closely related to isolates from human infections, suggesting recent exchange of bacteria between humans and monkeys. Our results are of public health importance, considering the increasing contact between humans and wild primates.
-
-
-
Replication kinetics of a CHIKV Asian strain with 76 aa duplication in the nsP3 HVD, in comparison to the wild-type Asian and ECSA strains
Introduction: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes fever, rash and polyarthralgia. CHIKV has expanded its circulating regions to the Indian Ocean islands, Europe, Americas and Southeast Asia. Two CHIKV lineages, the ASIAN and ECSA are circulating in Malaysia. In 2009, a CHIKV strain with a 76 amino acid (aa) duplication in its nsP3 hypervariable domain (HVD), identified as CHIKvASIAN09MUM-Dup+ was isolated from a patient co-infected with DENV-2. Indels and duplication have been found in many other alphaviruses, and suggested to play a role in the survivality of the viruses.
Objectives: We aim to compare and relate the replication kinetics and virulency in-vitro of CHIKvASIAN09MUM-Dup+ with the wild-type Asian and ECSA strains.
Methods & Results: Genotypic analysis was conducted on three CHIKV strains in Malaysia, the CHIKvASIAN06UM-Dup-, CHIKvECSA08UM-Dup- and CHIKvASIAN09MUM-Dup+. We found that CHIKvASIAN09MUM-Dup+ has significant low replication rates in Vero, C6/36 and Rhabdosarcoma cells as compared to the wild-type strains. The highest titers were reached by CHIKvASIAN09MUM-Dup+ in all cells are 6.5 to 6.75 log10 TCID50/mL, which is 100 fold lower compared to the wild-type strains.
Conclusion: The significantly low replication rate of Dup+ strain in all the cells, maybe suggestive to be due to co-infection and co-existence with DENV, where the aa duplication may play a role in overcoming competitive suppression. This preliminary finding agrees with reported events, where alphaviruses use insertion, deletion and duplication of amino acid in nsP3 HVD as strategies to influence replication in host, viral virulency, pathogenesis and survivality for evolution adaptation.
-
-
-
Developing global whole cell standards for the microbiome field
More LessThe microbiome field has developed rapidly over the last decade, however there is no effective standardisation of protocols in place, with no accredited or certified reference materials available to the wider community. As part of the NIBSC Microbiome program, we developed whole cell standards for microbiome research which can act as global working standards and will be put forward for consideration as the first whole cell World Health Organization International Reference Reagents for microbiome analysis. The developed reagents consist of common bacteria found in human gut and have been tested using different commercial DNA extraction kits in order to detect biases introduced through different DNA extraction processes. Multiple replicates of DNA extracted the whole cell standard using different DNA extraction kits were assessed using Next Generation Sequencing and evaluated using the number of input cells in the standards, as measured by microscopy and flow cytometry. These NIBSC whole cell standards are aimed to standardise the DNA extraction steps in microbiome analytical pipelines and serve as a tool to more accurately capture the representation of each microbe in the gut microbiome. This could be achieved by setting a specific threshold of accepted error levels by the microbiome community and we aim to set this through a large collaborative study in 2020-21. This work is part of a larger NIBSC microbiome program that is developing site specific DNA, whole cell and matrix spike-in reagents for use in microbiome research.
-
-
-
Regulation of Rhodobacter capsulatus gene transfer agent production
More LessHorizontal gene transfer (HGT) enables the spread of antimicrobial resistance, virulence, metabolic and other genes conferring an advantage to the organism. HGT is enhanced in biofilms because of increased cell-cell contact (conjugation), and eDNA in the biofilm matrix causing development of competence and providing material for transformation. Production of the Rhodobacter capsulatus gene transfer agent (RcGTA), another mechanism of HGT, could also increase in biofilm as high cell density increases the proportion of GTA particles produced that encounter a target cell. RcGTA is a phage-like particle that packages ∼4.5 kb pieces of random DNA from the producing cell’s genome and transfers it to a recipient cell. Five loci comprise the RcGTA genome: a 15kb cluster containing most of the RcGTA structural genes, a cell lysis locus, two structural loci encoding head spikes and tail fibres, and a maturation/regulation locus that includes that master regulator, gafA.
I assayed RcGTA production using gene transfer bioassays and biofilm using a 96 well plate assay. I will present data showing that deletion of four GTA-related genes, including gafA itself, all lead to reduced biofilm production. All four gene knock-outs also strongly reduce GTA-mediated gene transfer, suggesting GTA production and biofilm are co-regulated. I will also present work to characterize GTA production in biofilms, for example by monitoring the transfer of fluorescent protein genes through confocal microscopy, and assessing how specific regulators control this process. Biofilms are ubiquitous in the environment so studying the spread of antimicrobial resistance genes by GTAs is important.
-
-
-
Identifying drivers of microbial community composition associated with aquatic primary producers: metagenomic analysis of cyanobacterial cultures
More LessThe energy derived from aquatic primary production is fundamental to driving Earth’s life support systems – but they don’t achieve all this by themselves. Heterotrophic bacteria found in the photic zones of aquatic environments have a fundamental role in this too. Our group’s interest is in understanding how heterotrophs help autotrophs: who in these communities are important, and how and why they are important. Answering this is important in both natural and manmade environments so we can model these environments, and as appropriate, manipulate them, such as applying designer microbiomes to aid industrialisation of algal cultivation. Metagenomic analysis of 31 marine and freshwater cyanobacterial cultures from the Culture Collection of Algae & Protozoa resulted in assembly of >400 bacterial metagenomes (MAGs) with ca. 14 unique MAGs per culture. Community composition was clearly partitioned by salinity as a driver but collectively niche accounted for most community taxonomic variation. No universal core microbiome was identified, but taxonomic composition of marine cultures bore notable similarities to marine eukaryotic algal communities and to a natural cyanobacterial mat community found next to a northern Chilean geyser. Stable taxonomic associations imply that these taxa may have functional importance to their algal host. Functional analysis of the MAGs is underway and we will test whether the relative taxonomic variability contrasts with low functional variation between communities. If true, this implies that primary producers drive community assembly in a functionally predictable way, and that function, not taxonomy, is the more important parameter to understand.
-
-
-
The investigation of virus-driven intrahepatic cholangiocarcinoma through dysregulated hepatic differentiation
More LessIntrahepatic Cholangiocarcinoma (iCCA), bile duct cancer, is increasing in incidence worldwide. Infection with hepatitis C virus (HCV) is a known risk factor for developing iCCA. Recent studies of HCV associated hepatocellular carcinoma (HCC) have demonstrated that HCV infection induces oncogenic gene expression patterns resulting from altered epigenetic profiles. Importantly, whilst ∼90% of patients treated with new direct acting antivirals (DAAs) attain a sustained virological response, the risk of HCC and other malignancies is not reduced by the same extent as previous interferon therapies. We, and others, have shown that this may be related to an imprinted gene expression pattern persisting post-treatment.
The cellular origin of primary liver cancers can vary due to liver cell plasticity. However, the incidence of mixed iCCA-HCC tumour phenotypes and the dual iCCA/HCC risk associated with HCV infection led us to hypothesise that virus-infected hepatic progenitor cells (HPC) might be the source of virus-driven malignancies. Accordingly, we demonstrated that adult HPCs were susceptible to HCV infection ex vivo. Moreover, models of hepatic differentiation revealed that HCV disrupts this process via hijacking the HIPPO signalling pathway with oncogenic hallmarks persisting following viral cure.
To explore HCV-induced alterations during cholangiocyte-specific differentiation, we have chosen to exploit the immortalised non-transformed HPC line, HepaRG and induced pluripotent stem cell derived HPCs. We will describe endeavours to develop a robust model of HCV-mediated perturbation of cholangiocyte-specific differentiation in order to identify new treatments that complement DAA therapy in order to eliminate the risk of malignancy.
-
-
-
Gambian poultry isolates from hyperendemic group of AMR Escherichia coli strains in sub-Saharan Africa
Chickens and guinea fowl are commonly reared in Gambian homes as affordable sources of protein. Using standard microbiological techniques, we obtained 68 caecal isolates of Escherichia coli from ten chickens and nine guinea fowl in rural Gambia. After Illumina whole-genome sequencing, 28 sequence types were detected in the isolates (four of them novel), of which ST155 was the most common (22/68, 32%). These strains span four of the eight main phylogroups of E. coli, with phylogroups B1 and A being most prevalent. Nearly a third of the isolates harboured at least one antimicrobial resistance gene, while most of the ST155 isolates (14/22, 64%) encoded resistance to ≥3 classes of clinically relevant antibiotics, as well as putative virulence factors, suggesting pathogenic potential in humans. Furthermore, hierarchical clustering revealed that several Gambian poultry strains were closely related to isolates from humans. Although the ST155 lineage is common in poultry from Africa and South America, the Gambian ST155 isolates sit within a tight genomic cluster (100 alleles difference) of strains from poultry and livestock in sub-Saharan Africa (the Gambia, Uganda and Kenya). Continued surveillance of E. coli and other potential pathogens in rural backyard poultry from sub-Saharan Africa is warranted.
-
-
-
Investigating the impact of M1 protein from Group A Streptococcus on fibrin clot formation, structure and fibrinolytic potential
Background: Fibrin formation is an essential part of innate immunity, sealing off infections to limit bacterial spreading.The surface-anchored M1 protein is a major virulence determinant of Group A streptococcus (GAS). During early infection M1 is cleaved from the cell surface by SpeB, a streptococcal protease regulated by CovR/S. M1 forms a supramolecular complex with fibrinogen however the impact on fibrin formation is not known.
Methods: The effects of recombinant M1 (rM1) were assessed in fibrin clots made from plasma or purified fibrinogen incubated with thrombin, by confocal microscopy and scanning electron microscopy. Clotting and lysis profiles (with plasminogen activators and plasminogen) were investigated kinetically using thromboelastography (ROTEM).
Results: rM1 (0.47-60 μg/ml) increased clotting rates to produce heterogeneous clots with irregular fibre bundles and compacted fibrin. Formation of the protective fibrin biofilm was also disrupted by rM1. Furthermore, mechanical strength of fibrin clots was reduced with increasing rM1 concentrations and was undetectable above 15.5 μg/ml. Purified and plasma clots formed with rM1 were more susceptible to lysis by plasmin, with a 1.4 – 2-fold reduction in lysis times.
Conclusions: At sites of GAS infection, cleaved M1 may bind to fibrinogen generating fibrin clots which: lack the protective film at the clot surface; are mechanically weaker; and are less resistant to lysis by plasmin. GAS strains of M1-type are commonly associated with invasive infections; the impact of M1 on fibrin structure could contribute to the severity of GAS infection by compromising the fibrin barrier that limits bacterial proliferation and migration.
-
-
-
Isolation of thermophilic Actinobacteria from compost and identification of bioactive compounds with antimicrobial properties
More LessTo combat the problem of antimicrobial resistance, we are testing the hypothesis that thermophilic Actinobacteria produce novel antimicrobials at higher temperatures, with potential activity against life-threatening infections like invasive aspergillosis caused by the fungus Aspergillus fumigatus. Samples from “windrows” at a green waste processing facility yielded 36 potential thermophilic Actinobacterial strains isolated at 50oC, as well as strains of A. fumigatus. The phylogeny and identities of the bacterial strains were determined by 16S rDNA sequencing. Three strains - DJT 15 Streptomyces thermoviolaceus subsp. apingens, DJT 32 Saccharomonospora viridis and DJT 36 Saccharomonospora glauca - have shown inhibitory activity in bioassays against the ESKAPE pathogens, two of which (DJT 32 and 36) also inhibited the growth of the fungal pathogen Aspergillus fumigatus isolated from the same compost. Strain DJT 32 has also been shown to have an inhibitory effect against azole resistant human pathogenic strains of A. fumigatus. Whole genome Sequencing data of DJT 15 and 32 have been used to identify possible biosynthetic gene clusters for antimicrobial compounds (novel or otherwise) through AntiSmash analysis. Alongside this, bioactive compounds have been extracted from broth cultures of each strain using HP-20 Resin method, and the metabolites will be identified using LC:MS combined with metabolic profiling. Extraction and identification of novel metabolites will provide a path for the development of new antimicrobials for clinical use. This study has shown that thermophilic Actinobacteria produce antimicrobial compounds at higher temperatures, against Staphylococcus aureus and against the highly pathogenic fungus, A. fumigatus.
-
-
-
Anoxygenic phototroph pufLM gene sequences derived from tropical aquatic sampling sites: Diversity, distribution, and phylogenetics
More LessPurple sulfur bacteria (PSB) and purple non-sulfur bacteria (PNSB) are characterized by their ability to perform anoxygenic photosynthesis. PSB and PNSB are ubiquitously found in coastal waters, enclosed lagoons, stagnant water, mangrove soils, estuaries, and similar environments. In this study, we examine microbial diversity in PSB enrichments derived from a variety of tropical sampling sites (e.g., Thailand, Puerto Rico) associated with shrimp ponds, coastal mangroves, fresh water ponds, and Nymphaeaceae (i.e., water lily) plant tissue. Since 16S rRNA-based analyses are inadequate to describe the diversity of phototrophic bacteria, other biomarkers (e.g., pufLM) are used to construct phylogenies and elucidate biogeography. Our samples indicate that the majority of sequences associated with freshwater pond PSB were related to known marine, halophilic, or salt-tolerant PSB (e.g., Marichromatium, Allochromatium, Thiococcus, and Thiohalocapsa). Phylotypes not closely-associated with known species of PSB (or PNSB) were also found. PNSB gene sequences, which appear to be related to Rhodopseudomonas and Rhodoplanes, were mostly found in freshwater samples and from Nymphaeaceae plant tissues, suggesting a difference in the ecology and distribution of these two broader bacterial groups. This difference is likely due to differences in habitat such as physical (e.g., temperature) and chemical parameters (e.g., salinity). Our preliminary analyses demonstrate a rich diversity of anoxygenic phototrophic bacteria from tropical sampling sites. Few studies have described the diversity of purple bacteria in tropical environments using full pufLM gene sequences. Employing next-generation sequencing (NGS) appears to provide greater resolution towards a deeper understanding of the global diversity and distribution of these anoxygenic phototrophs.
-
-
-
Isolation and characterization of Anoxygenic phototrophs from shrimp ponds in Thailand
More LessAnoxygenic phototrophic purple bacteria are ubiquitous in aquatic and terrestrial environments and demonstrate broad phenotypic diversity. Purple bacteriaderive energy from light under anaerobic conditions via anoxygenic photosynthesis, a process in which water is not the electron donor. It has been suggested that these bacteria are useful for a variety of applications, including: wastewater treatment; heavy metal remediation; nitrogen fixation; and, control of CH4 emissions. In this study, the goal was to isolate and characterize PNSB from shrimp ponds in Thailand. Surface water and sediment were collected. Enrichment cultures were prepared using Pfenning’s mineral media. As indicated by development of reddish color and turbidity, anoxygenic phototrophic growth was observed within two days of incubation. Cultures in liquid media and on solid plates exhibited a deep red or purple color ten weeks post-inoculation. Under light microscopy, enrichments consist of communities dominated by thin, elongated gram-negative cells with granules of elemental sulfur, which are characteristic of purple bacteria. Molecular methods confirm the presence of pufLM, a genetic biomarker for purple bacteria (e.g., Thiohalocapsa marina, Allochromatium vinosum, Roseovarius tolerans). Initial sequencing of key genes (i.e., pufLM) indicate that these environmental samples contain novel isolates or “geographic variants” that have not been previously described. We have developed a few pure cultures of multiple species from these environmental samples. Since shrimp farming is a key industry in southern Thailand, the characterization of the microbial communities in these ecosystems, including anoxygenic phototrophs, will provide insights into how to maintain water quality in these food production systems.
-
-
-
Investigating the functional relationship between streptokinase variants from Group A Streptococcus, and associated M-like proteins
More LessBackground: Streptokinase (SK) from Group A streptococcus (GAS) activates human plasminogen to generate plasmin, which degrades fibrin clots tofacilitate bacterial dissemination. Sequence variants of SK from diverse GAS strains form distinct evolutionary clusters.Unlike cluster 1 SK, cluster 2 variants have very little activity in solution and depend on co-factors (e.g. fibrin(ogen)).Cluster 2 SK variants also appear to correlate with cell-surface M-like proteins: SK2a with M1 (fibrinogen-binding) and SK2b with PAM (plasminogen-binding).
Methods: Plasminogen activation by recombinant SKs (rSK2a, rSK2b) was investigated by chromogenic assay; nickel-coated microtiter plates were used to immobilise recombinant M proteins (rPAM and rM1) via a C–terminal His tag to mimic cell surface plasmin generation.
Results: Plasminogen activation by rSK2b is stimulated ∼18-fold by rPAM in solution; when rPAM is immobilised stimulation exceeds 100-fold. Fibrin is the most potent stimulator of rSK2a activity (7-fold increase) compared to fibrinogen (4-fold); when rM1 was included, either in solution or immobilised, there was no further stimulation of rSK2a activity with fibrinogen.
Discussion: Stimulation ofSK2b activity by plasminogen bound to immobilised PAM suggests an important role for cell-surface plasmin generation. SK2a activity appears to be independent of M1, targeting fibrin directly. SK variants are commonly associated with distinct disease manifestations with SK2b commonly expressed by invasive skin-tropic strains of GAS and SK2a by nasopharynx-tropic strains. An improved understanding of the molecular mechanism of action by GAS SK variants may help to identify potential novel therapeutic targets for the treatment of invasive GAS diseases.
-
-
-
Synergistic degradation of a biodegradable plastic film by a marine microbial community
More LessBackground: The need for replacing conventional plastics has led to an increase of the use biodegradable plastics. Most biodegradable plastic materials are certified for compostability, and their degradation mechanisms by marine bacterial communities, is still largely unknown.
Methods: Bacterial communities that degrade a PBAT-based biodegradable film (PF) were enriched from marine samples collected from the Mediterranean (Greece and Italy) and North Sea (Germany). DNA, RNA and proteins were extracted simultaneously from cultures that were starved and induced with biodegradable films, for metagenomic, transcriptomic and proteomic analyses. Mineralization of the films was assessed by CO2 evolution and film disintegration was physicochemically assessed.
Results: Within the enriched marine communities, Alphaproteobacteria and Gammaproteobacteria were the most abundant classes. Among these groups, Marinobacter was the most predominant genus on the PF film. Hydrolases similar to PETases, MHETases and terephthalic acid dioxygenases, the enzymes needed for polyethylene terephthalate (PET) degradation, were expressed when communities were exposed to the biodegradable PF film. The PETases-like hydrolases (Ple) belonged to Marinobacter, MHETases-like hydrolases (Mle) to Marinobacter and Pseudooceanicola species, and the putative terephtalate dioxygenases (Tph) to Saccharospirillum and to a α-proteobacterium candidate. Ple proteins were mainly abundant and upregulated on the PF film while Mle and Tph were abundant in the free-living fraction. Around 60% of the tested biodegradable plastic was converted to CO2 and no traces of the film were detected after the mineralization was complete.
Conclusion: Biodegradable plastics degradation is achieved synergistically by labour division among specialized film-attached and free-living bacteria. Ultimately, their complex interaction leads to the complete mineralization of a biodegradable plastic.
-
-
-
Impact of Amoxicillin treatment on E. coli in dairy cow faeces
More LessIntroduction: There has been a global drive for increased antimicrobial stewardship. In the UK, this drive has focused on decreased usage of antimicrobials, specifically HP-CIAs. It is well known that antimicrobials are selection drivers for antimicrobial resistance. How a given dose will impact the prevalence of resistance in a microbiome, and therefore the associated risk of a resistant infection is less understood. Questions also remain around the impact of antimicrobial therapy on resistance across a herd, if selection occurs within an animal receiving treatment, what is the potential risk of this resistance spreading throughout other animals?
Methods: E. COLI ISOLATION Tryptone Bile X-Glucuronide (TBX) media was used for selective growth of non – toxigenic E. coli from dairy cow faeces. SUSCEPTIBILITY TESTING EUCAST Disk Diffusion testing guidelines were followed to determine susceptibility of faecal isolates to a panel of 8 antimicrobials from 5 different classes. ISOLATE FINGERPRINTING To determine clonality of faecal isolates ERIC-PCR was used as an efficient method to provide a genomic fingerprint.
Results This work is ongoing. Current work suggests that low frequency Amoxicillin treatment has no significant selection for resistance. However, there appears to be some instances of co-selection for Streptomycin, Tetracycline and Sulphonamide resistance, and several multi drug resistance isolates have been identified. More work needs to be done to confirm the impact of low frequency Amoxicillin treatment on E. coli resistance and identify the mechanism behind suspected co-selection and multi drug resistance.
-
-
-
Human host cell entry restriction of Lassa and other arenaviruses
More LessArenaviruses are the largest family of viral haemorrhagic fever causing viruses. They have worldwide distribution and are divided into Old World (OW) and New World (NW) viruses based on their phylogeny, geographical distribution and serological cross-reactivity. Endemic to West Africa and South America, these emerging RNA viruses jump the species barrier from their natural rodent hosts to humans, resulting in illnesses ranging from mild flu-like syndromes to severe and highly fatal haemorrhagic zoonoses. Recent increased frequency of outbreaks and associated high fatality rates of the most common arenavirus, Lassa, in Nigeria has emphasised that these viruses should no longer be treated as causes of sporadic epidemics. The immense impact of these outbreaks on human health is further exacerbated by the lack of vaccines and effective treatments and makes it imperative to understand the molecular basis of viral pathogenesis and immune evasion.
Virus entry is a key determinant of viral host range, cellular tropism and disease outcome, hence, targeting this step of the arenavirus lifecycle could have significant impact on the control of viral infection. Our data demonstrate for the first time a synergistic restriction activity against arenavirus entry by two cellular host factors known for their control of enveloped virus infections. This co-operative restriction activity appears to conserved and we have evidence that arenaviruses may have evolved strategies to escape inhibition, through entry receptor switching, thus alluding to an understanding of the dynamics of arenavirus infection and adaptations that the viruses have made to escape host restriction pressures.
-
-
-
Structural investigation of cyclic nucleotide binding proteins from Trypanosoma cruzi
More LessFora targeted therapy of Trypanosomiasis, new antiparasitic drugs should be specifically directed against essential pathways in the parasite life cycle. Among these potential targets are signal transduction pathways, which have remained largely unexplored in Trypanosoma species. Of special interest is cAMP-mediated signaling, since cAMP has been shown to play critical roles in the life cycle of T. cruzi and in host cell during invasion. The presented research focuses on the identification and characterisation of novel cAMP response proteins (CARPs) in T. cruzi by using a multi disciplinary approach involving the parasitology group of Dr Martin Edreira (University of Buenos Aires, Argentina) and the structural biology group of Dr Ivan Campeotto (University of Leicester, UK). The aim of the project is not only to increase our knowledge about T. cruzi biology but also to target CARPs for the design and development of novel therapeutic agents against Chagas disease. To date, protein crystals of one of the members of the CARP family have been obtained, paving the way for structure determination and for a structure-based drug design approach.
-
-
-
Diverse mutational routes to the restoration of motility in a soil bacterium
More LessRestoration of a phenotype following insertion of a crippling mutation gives us insights into the adaptability of an organism. Mutational routes to the restoration of phenotypes can be diverse. Deletion of the master regulator (fleQ) for flagellar synthesis genes renders Pseudomonas fluorescens immotile, but after being put under strong selection to swim, it reliably re-evolves motility.
In order to uncover how the immotile bacteria regain their ability to swim they are inoculated into swimming agar plates and sampled from the leading edge of the motile zone. These samples are sequenced at a range single locus sites, predicted to be where causative mutations for the restoration of motility will be found (as indicated from previous research).
In the engineered immotile strain Pf0-2x, a huge variety of mutations, consisting of SNPs, multi-codon deletions, and frameshifts are observed across all candidate loci. In a complex environment (LB) 50% of initial restorative mutations occur in glnA, a glutamine synthetase gene. In a minimal media environment (M9) 45% of these mutations occur in ntrB, a gene for a kinase that forms part of a two component system. These findings contrast strongly with similar work in the related P. fluorescens strain SBW25, where the same SNP is repeatedly found across all environments. Interestingly, this SNP has never been seen in Pf0-2x.
These results highlight how preferential mutational routes can vary across environments, and that strain-to-strain differences can have a major impact types of mutations that are uncovered and selected for by evolution.
-
-
-
Genomic epidemiology of Campylobacter jejuni associated with asymptomatic pediatric infection in the Peruvian Amazon
More LessCampylobacter is the leading bacterial cause of gastroenteritis worldwide and its incidence is especially high in low- and middle-income countries (LMIC). Disease epidemiology in LMICs is different compared to high income countries like the USA or in Europe. Children in LMICs commonly have repeated and chronic infections even in the absence of symptoms, which can lead to deficits in early childhood development. In this study, we sequenced and characterized C. jejuni (n=62) from a longitudinal cohort study of children under the age of 5 with and without diarrheal symptoms, and contextualized them within a global C. jejuni genome collection. Epidemiological differences in disease presentation were reflected in the genomes, specifically by the absence of some of the most common global disease-causing lineages. As in many other countries, poultry-associated strains were a major source of human infection but almost half of local disease cases (15 of 31) were attributable to genotypes that are rare outside of Peru. Asymptomatic infection was not limited to a single (or few) human adapted lineages but resulted from phylogenetically divergent strains suggesting an important role for host factors in the cryptic epidemiology of campylobacteriosis in LMICs. Preprint: https://www.medrxiv.org/content/10.1101/2020.04.26.20075689v1
-
-
-
Antimicrobial resistance: Transdisciplinary research on humans, antimicrobials and microbes
More LessCalls for action on antimicrobial resistance (AMR) have existed almost as early as the discovery of penicillin and the sulpha drugs. Since then solutions to AMR have circled around the development of new antimicrobials and the rationalisation of their use via various configurations of regulation of access and distribution, promotion of diagnostics and education of prescribers and consumers. Research by historians and social scientists (HSS) are increasingly demonstrating the various limitations and unintended consequences of many of these approaches, while also seeking to propose not only different ways to study AMR as a problem, but also address it. Part of this, involves serious engagement with microbiological insights (i.e. related to the microbiome) and methods to move beyond the impasses of outdated concepts (e.g. germ theory), methodological reductionism and disciplinary boundaries. Based on our empirical research on AMR and human microbiome science, we demonstrate how AMR is a transdisciplinary problem requiring contributions from HSS’s research and expertise in order to devise socially meaningful and microbiologically effective solutions. We have identified four areas where such contributions would be beneficial: (1) policies (e.g. AMR policies still assume germ theory and operate within silos); (2) AMR solutions are human centred (i.e. neglect of the microbiome and pay limited attention to other nonhumans) vs one health; (3) epidemiological variables and microbiological discourse (i.e. often employ outdated anthropological and philosophical concepts, such as westernised, modern, traditional); (4) Rhetorics and lexicon (i.e. can be morally and conceptually simplistic, like ‘war’, ‘sweets’, ‘good’/’bad’ bugs, ‘irrational’).
-
-
-
Molecular identification of antibiotic resistance genes of bacteria isolates from ready to eat food and drinking water sold in Imo State University
More LessFood and water are fundamental need of human as its quality is of great concern to the generality of consumers, despite quality control measures during their production, unacceptable microbiological load often makes them unsafe. In this study, we analyzed various ready to eat food and water for presence of coliform bacteria, fungi count, antibiotic resistance gene and plasmid DNA, Samples were found to be positive for various antibiotic resistance genes namely, Sul1, Sul2, Tet A and TetB, interestingly, considerable total coliform bacteria were found from isolates and this result was further confirmed using illumina sequencing of the 16SrRNA gene. All Samples were negative for plasmid DNA, These finding deserve attention as the presence of coliform bacterial and antibiotic resistance genes potentiate health risk to members of university community and as such calls for strigent supervision and saftety and implementation of food safety regulations.
-
-
-
Characterization of a multi-modular bacterial enzyme with binding and hydrolysis activities on fungal cell wall components: Insights into bacterial-fungal interactions in the soil
More LessBacteria from the phylum Bacteroidetes is known as good degraders because of their abilities in secreting glycoside hydrolases (GHs). As a member in Bacteroidetes, Chitinophaga pinensis is capable to hydrolyze several glycans (McKee et al., 2019). We found that a multi-modular CAZyme produced by C. pinensis contains not only two GHs but also two uncharacterized domains. Although it is common for CAZmes to have non-catalytic modules like carbohydrate-binding domains (CBMs), the domains in this protein are uncommon, and their functions unknown.
Our enzyme contains five domains – a GH5_46 enzyme, a GH18 enzyme, two non-identical uncharacterized domains, and a Por C-terminal secretion domain. I have shown that the GH5 can specifically degrade pustulan (β-1,6-glucan), and is the only β-1,6-glucanase found in its sub-family. The GH18 is a chitinase that reacts with β-chitin. Interestingly, the two uncharacterized domains both bind polysaccharides, and improve enzyme stability. However, they do not necessarily bind the same glycan as their GH partners. Our result demonstrates how two distinct polysaccharide-degrading modules within one protein can cooperate. We also discuss the impact of the binding domains on the GH activity.
We anticipate our research can stimulate the exploration of β-1,6-glucanase activity in family GH5, and expand the database of carbohydrate specificities in bacteria. The two unusual binding domains we have characterized suggest an unexplored aspect of bacterial physiology in the soil.
References:
McKee, L.S., et al., 2019. Focused metabolism of β-Glucans by the soil Bacteroidetes species Chitinophaga pinensis. Appl. Environ. Microbiol., 85(2), pp.e02231-18.
-
-
-
Using structural biology of methanogen enzymes to aid our understanding of methane formation
More LessMethane is a potent greenhouse gas (28-fold more potent than carbon dioxide) and is a significant gas contributing to global climate change. Approximately a billion tons of methane are produced each year by methanogenic archaea in ruminants. These archaea possess a number of unusual traits such as isoprenoid-based lipids, unusual cell wall chemistry and a unique energy metabolism (methanogenesis) that requires six methanogen-specific cofactors. Many of the enzymes involved in these processes have no direct analogues in the host animal. To gain insights into the fundamental biology of rumen methanogens we have determined crystal structures of key enzymes with archaeal-specific traits. Over 600 enzymes were targeted for structure determination which produced approximately 200 purified soluble enzymes for crystallographic screening. More than 50 different enzymes have produced crystals and 30 structures have been solved for individual enzymes to date. The results have helped illuminate our understanding of methane formation at this critical juncture in the world’s history.
-
-
-
Can m6A-modified RNA be used as an Anti-Viral Target?
More LessKaposi’s sarcoma-associated herpesvirus (KSHV) is a DNA virus associated with several HIV-associated malignancies.
Like all herpesviruses, KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND-1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019).
Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner. Taking this into consideration, novel ligands have been assessed as effective anti-viral reagents. The importance of A versus m6A within the lytic phase of KSHV’s lifecycle will be investigated by combining in silicoscreening with biophysical techniques and cell based assays.
-
-
-
Identification of potential key genetic factors in the long-term success of Shigella as pathogens
More LessBacterial of the genus Shigella are a major contributor to the global diarrhoea burden causing 100,000 deaths per annum globally. Further to this, increasing antibiotic resistance in Shigella and the lack of a licenced vaccine has led WHO to recognise Shigella as a priority organism for the development of new antibiotics. Understanding what drives the long-term persistence and success of this pathogen will thus aid the global management of shigellosis and may identify targets relevant to other enteric bacteria. To identify key genetic drivers of Shigella evolution over the past 100 years, we have used the historical Murray collection; comprising several hundred pre-antibiotic era (1917 – 1954) Enterobacteriaceae from diverse geographical locations. We employed genome-wide association studies to sequences from over 100 Shigella isolates from the Murray collection alongsidemore modern (i.e. 1950s – 2018) isolates to identify genetic factors (SNPs and genes) significantly associated with time as a continuous variable. GWAS hits (e.g. a putative resistance protein) then underwent variation, functional and phenotypic analysis to examine the plausibility of their role in shaping Shigella populations and as potential targets for managing this pathogen.
-
-
-
Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Despite on-going vaccination programmes, Neisseria meningitidis causes over 700 cases of invasive meningococcal disease (IMD) in the UK each year. In 2017-18, the MenW and MenY capsular groups caused 38% of all IMD cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to understand how genetic variation contributes to phenotypic differences between carriage and disease isolates.
We are adapting a variety of assays, designed to mimic carriage and disease behaviours, for high throughput phenotypic testing of multiple meningococcal isolates from carriage and cases of IMD. We have selected 335 MenW cc11 and MenY cc23 isolates and are currently testing subsets of isolates in cell culture (CaLu3), growth and biofilm assays. Phenotypic differences will be utilised as input data for Genome Wide Association Studies that aim to identify the specific genomic variants, or combinations of variants, determining observed differences. Genomic data will include whole genome sequences and repeat-mediated phase variation states.
Our preliminary data has detected variation in the ability of cc11 and cc23 isolates to disrupt monolayers of CaLu3 cells, indicating that minor genetic differences in phylogentically similar organisms may be physiologically important for both carriage and disease. We will also discuss progress in establishing successful, high-throughput assays for testing multiple isolates.
-
-
-
Digital Outreach: Promoting a global understanding of antimicrobial resistance (AMR)
More LessTechnology allows educators to reach a global audience without amassing a large carbon footprint. The Skype in the Classroom programme from Microsoft enables me to spark curiosity and engage a wide range of students in dialogue to explore global issues, in particular, the threat of antimicrobial resistance, while supporting me to develop my skills as an educator. Through a multi-disciplinary approach, I employ Skype in the Classroom as a future-proof, powerful tool in our arsenal against the evolving microbial threat. We are not only responsible for teaching the next generation of scientists, but also the next generation of thought-leaders and professionals, who will work collaboratively towards our common goals.
Antimicrobial resistance is one of the grand challenges of the 21st century, and global connectivity is one of the best solutions to promoting better understanding of this threat. The Skype in the Classroom programme has allowed me to teach sessions to students in Norway, India, Brazil, Miami, Florida and Puerto Rico, with students ranging from 10 to 18 years old. I use skype and Teams to deliver online lessons in an accessible way, starting with the scope of the problem, covering the technical detail of how resistance emerges and is spread, and looking at some of the newest approaches to tackling AMR. Using a ‘challenge-based-learning’ approach, we discuss how different professionals and roles within society can contribute to the issue. This makes the class applicable to students with a wide range of backgrounds, including those without a keen interest in science.
-