- Volume 2, Issue 7A, 2020

Phenotypic 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.

Non-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.

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.

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.

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.

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.

The 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.

Horizontal 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.

The 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.

Intrahepatic 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.

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.

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.

To 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.

Purple 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.

Anoxygenic 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.

Background: 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.

Background: 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.

Introduction: 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.

Arenaviruses 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.