- Volume 2, Issue 7A, 2020

Bovine rotavirus (RV) infection causes severe diarrhoea in young dairy calves and has a significant economic impact on livestock production as a result of high morbidity and mortality caused. Development of technologies to engineer infectious RV using an entirely plasmid-based reverse genetics (RG) system has proven challenging. A breakthrough was made when Kanaiand co-authors (PNAS, 2017)developed a plasmid-only-based RG system for the simian RV strain SA11.We are currently developing an analogous RG system for the bovine RF RV strain. Having parallel systems for different RV strains will help to validate phenotypic changes induced by site-directed mutagenesis (SDM) within the RV genome.

The coding capacity of the 11-segmented dsRNA RV genome has been largely unexplored. Using bioinformatic analyses, we have identified four segments with up to five putative alternative initiation codons which are in moderate or strong Kozak context. Furthermore, some occur in segments for which the canonical start codon occurs within 15 nucleotides of the start of the segment, further suggesting the possibility of alternative translation start sites to generate coding diversity. We are now applying our RG systems to investigate RV coding capacity using TnT transcription assays, radiolabelling and SDM.

Diphtheria is a potentially life-threatening infection in humans. The three species capable of causing diphtheria are: Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Although UK cases are rare, recently there has been an increase in reported toxigenic C. ulceransassociated with companion animals. Potentially toxigenic corynebacteria are sent to the National Reference Laboratory, Public Health England (PHE), London, UK for species confirmation, determination of presence of the diphtheria toxin gene by real-time PCR, and confirmation of toxin expression by the Elek test. We reviewed submissions of C. ulcerans between January 2006 and November 2019.

Fifty-three isolates of toxigenic C. ulcerans were received, 29 from humans and 24 from animals. The most common animal hosts were dogs (15) and cats (7), but isolates were also received from a horse and a captive rhinoceros. Multi-locus sequence typing (MLST) data were derived from whole genome sequencing. In three human cases, C. ulcerans was isolated from companion animals and in all three, typing data supported an epidemiological link between human and animal hosts. The sequence types (STs) for these linked isolates were ST331 (human and dog, human and cat) and ST551 (human and cat).

Although MLST data are limited, the finding of the same ST (ST331) from canine and feline sources indicates that strains can be carried by both hosts.

Management of diphtheria cases is an important public health issue. Typing data can support epidemiological linkage and identify possible sources, allowing the potential intervention and treatment of animals thus avoiding onward transmission.

Trimethylamine N-oxide (TMAO) is an osmolyte that enters the bloodstream directly through consumption of fish, or through microbial metabolism of it and other dietary methylamines such as carnitine and choline, producing trimethylamine (TMA). TMA produced by microbes enters the blood and is transported to the liver, where it is converted back to TMAO by flavin-containing monooxygenases before being excreted in the urine. TMAO has been shown to potentially have beneficial effects on metabolic health and the gut-brain axis at normal physiological levels, while elevated serum levels are associated with cardiometabolic disease in Western patients. Bacteria in the family Enterobacteriaceae exhibit changes in their growth and metabolome in the presence of TMAO but why this occurs is not known. Growth experiments show that a caecal isolate of Klebsiella pneumoniae, a member of the Enterobacteriaceae, experience a more rapid growth rate, when grown anaerobically in the presence of TMAO, but may require oxygen to be present to produce TMA. K. pneumoniae will be subjected to differing concentrations of TMAO and oxygen to examine the effects on the metabolites produced, the genes expressed, and the rate of growth. This will be done by analysing spent media using GC-MS, qPCR targeting genes involved in anaerobic respiration, and measuring the turbidity of cultures over time. By understanding how bacteria in the gut interact with TMAO new insights can be gained as to how gut bacteria and their metabolites influence human health.

Influenza D virus (IDV) has been reported in many animal species and potentially humans worldwide. Cattle are considered the major reservoir. There are currently three main lineages based on the haemagglutinin-esterase (HEF) gene: D/OK, D/660 and D/Japan. We performed pilot surveillance for IDV by using pseudotyped lentivirus (PVs) to generate a cell-based test to identify prior-exposure to IDV in animals. The expression plasmids of the HEF genes, D/swine/Italy/2015, D/bovine/France/2014, and D/bovine/Ibaraki/2016, were constructed. The HEF plasmid was co-transfected with lentiviral vector plasmid expressing luciferase, lentiviral Gag-Pol plasmid, and HAT protease plasmid in producer cells (HEK293T/17). Three days post-transfection, supernatants were collected and used for titration on various cell lines and in micro-neutralisation tests. Sera from pigs vaccinated with D/swine/Italy/2015 and D/swine/Oklahoma/2011 were used to undertake a preliminary validation of the micro-neutralisation assay. All pig sera have neutralising activity to influenza D (Italy) pseudotyped lentiviruses. Cow and sheep sera, 145 and 114 specimens, respectively, collected from UK farms were screened using the micro-neutralisation test. We found 97 bovine sera (66.9%) were influenza D antibody positive. Collectively, pseudotyped lentivirus technology opens up opportunities for serological surveillance of influenza D viruses.

Despite being the number one cause of death from an infectious disease, little is known of the 7 phylogenetic lineages of Mycobacterium tuberculosis (Mtb). These lineages are thought to have adapted differently to their human hosts, as they are geographically localised. As a result, they show variation at the phenotype level, such as virulence and the ability to develop antibiotic resistance, and at a genomic level, such as in single nucleotide polymorphisms (SNPs). We have linked the differences in SNPs between lineages to differences in metabolites (i.e. what is ultimately produced by a cell). Through multi-omic integration of these datasets we have discovered lineage-specific metabolomic changes, potentially as a result of genomic adaptation. The differences between lineages will provide insight into new biological pathways to target and manipulate in future research.

Candida albicans is an opportunistic fungal pathogen present in the oral cavities of up to two-thirds of people. Despite typically existing as a commensal microorganism, it has pathogenic potential, particularly in older, immunocompromised individuals. A common Candida-associated infection is denture-associated stomatitis (DS), which presents clinically as areas of erythema on the palatal mucosa, and discomfort for the denture-wearer. In vitro, previous work has shown that the expression of C. albicans virulence factors varies according to its interactions with other oral microorganisms.

Mature single- and mixed-species biofilms (with Candida and several strains of common oral bacteria) were grown on poly(methyl methacrylate) (PMMA) coupons, representing dentures. Additionally, to some coupons, individual probiotic strains were added. Total RNA was extracted, reverse transcribed and putative virulence gene expression was determined by RT-qPCR relative to ACT1, a housekeeping gene. Biofilm-infection assays of FADU and TR146 epithelial cell lines were also performed by pre-culturing cells, then adding single- or mixed-species inocula overnight. Quantification of cell damage determined by lactate dehydrogenase assay.

Biofilm co-culture with the addition of certain probiotic strains downregulated C. albicans virulence genes in both short-term and long-term mixed-species biofilms. With an increasing aged population that is heavily reliant on the use of antibiotics that can negatively affect the microbiota of patients, there is a requirement to look at the benefits of prophylactics, from both an economic and patient well-being viewpoint. The results show the realistic possibility of using probiotics to prevent or restrict development of Candida-associated oral diseases.

Gene transfer agents (GTAs) are small viruses that package and transfer random pieces of the producing cell’s genome but are unable to transfer all the genes required for their own production. GTAs are able to spread any DNA in the host cell and so their potential impact upon bacterial evolution and antimicrobial resistance is immense.

Our discovery that the product of gene rcc01865 is a specific GTA activation factor (GafA) for the model Rhodobacter capsulatus GTA (RcGTA) and that GafA is essential for RcGTA production, has provided the link between GTA production and host regulatory pathways. However, while GafA has significantly improved our understanding of GTA regulation the complete mechanism is unclear. Our goal was to investigate the GafA mechanism of action in more detail. We demonstrate direct protein-protein interaction between GafA and the RNA polymerase omega subunit (RNAP Ω) using bacterial-two-hybrid and pull down assays. Further evidence for the interaction has come from random and site directed mutagenesis of gafA and targeted truncations. GafA mutants were also tested to assess their impact on RcGTA production. RNAP Ω is thought to recruit alternative sigma factors to the RNAP holoenzyme. Regions of GafA also share sequence homology with known sigma factor proteins, and we propose that GafA acts as an alternative sigma factor to co-ordinate expression of disparate RcGTA genes. Our results advance our understanding of this fascinating mode of horizontal gene transfer, not only in the model species but also in other potential GTA producing species that contain gafA homologues.

Bumblebees play a major role in global pollination. Consequently, their health is of high importance for food security worldwide. Yet, recent population estimates show that their numbers are declining. This decline has been attributed to habitat loss, infection and use of pesticides. An important factor for bee health that contributes to population survival is the gut microbiome composition. The bee gut microbiome provides protection from pathogens, is specific to the host and helps break down food. Without a balanced gut microbiome, the health of the bee is threatened through increased infection and mortality. The bee gut microbiome is relatively simple, being dominated by 8 core bacterial species providing a convenient study system. Previous published data shows that air pollution has an impact on bacterial behaviour. Therefore, our hypothesis is exposure to air pollution causes an imbalance in the bee gut microbiome. To test this, we exposed bees to black carbon (BC), a major component of air pollution particulate matter. We assessed the effects on bee behaviour, microbiome composition and gut bacteria treated in vitro. Bees treated with BC showed a significant increase in viable bacterial cells in their faecal community. Independent culture of gut commensals showed that BC significantly alters the structure of their biofilms, which are important for colonisation in vivo. This supports the hypothesis that air pollution can cause an imbalance in the bee gut microbiome, and may adversely influence bee health and pollinator populations.

Cryptosporidium spp. are microbial parasites that infect the gastrointestinal tract of humans and many animals, causing cryptosporidiosis, a disease characterised by acute watery diarrhoea. In dairy farms, C. parvum is the most common species in calves, leading to high mortality rate, stunted growth, and consequently high economic losses. Trade between farms and breeding centres is a major risk factor in the spread of such parasites, posing a threat to other farms worldwide as well as to human health. This problem is aggravated by the lack of good breeding practices, efficient detection tools, and lack of effective anti-cryptosporidial drugs.

To address cryptosporidiosis in dairy farms, we have established the ‘Health For Dairy Cows (H4DC)’ consortium in order to tackle some of the aforementioned issues.

Herein, we will present preliminary data from a 3-step strategy:

1)Dissemination of pilot farms in France, Belgium, The Netherlands and England. This collaboration will serve to test the effect of new husbandry practices in the occurrence of Cryptosporidium, which will aim to decrease Cryptosporidium incidence and ultimately decrease the economic burden of cryptosporidiosis.

2)These pilot farms will later be used as testing-grounds of the low-cost and easy-to-use in-situ C. parvum detection tool that will be developed during this project.

3)Development of a cell-based drug-screening system which will be used to screen various drugs and compounds for anti-Cryptosporidium activity.

Finally, data from these findings will be used to establish model and strategies in order to transfer the developed technologies to both farmers and biotech/pharmaceutical companies.

The 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.

Microcin 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.

With 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.

Antimicrobial 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.

P. 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.

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.

Mycobacterium 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.

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.

The 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.

Abyssomicin 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.