- Volume 4, Issue 5, 2022

Human and animal pathogenic bacteria are constantly being released into the environment through human activity. Many of these organisms can harbour genes such as virulence genes, antibiotic resistance and heavy metal resistance genes that are inserted into plasmids, transposons and Integrating Conjugating elements (ICEs), leading to potential spread. Such spread can be detected among water and soil communities and in particular in wastewater treatment plants. This makes wastewater and treatment plants a potential reservoir for mobile genetic elements including SXT/R391 ICEs, commonly detected amongst enterobacterial genera. Many plasmid and ICE genomes have been detected serendipitously from clinical sources but few have been identified without selection. Here we examined a domestic wastewater treatment plant to identify, isolate and characterize SXT/R391 ICE’s without selection. Standard microbial replica plating in conjunction with ICE specific (conserved integrase gene) PCR techniques were employed to identify an SXT/R391 ICE MGE using a range of enterobactial selective media. A Novel SXT/R391 ICE MGE was identified from a wastewater Proteus mirabilisstrain. Whole genome sequencing using Ilumina sequencing technology revealed a novel 81 kb element which on annotation contained 75 open reading frames. The “hotspot regions”, which contain adaptive genes, encoded a novel bacteriophage defence mechanisms but lacked other selectable determinants. With the continuous arms race between bacteria and phage, bacteria have developed novel resistance mechanism systems that protect the bacteria from phage. Such systems may be key adaptive mechanisms harboured by ICEs particularly in wastewater systems which will contain large phage populations.

Antibiotic resistance is one of the biggest public health challenges of the 21st century. The Rtc RNA repair system is present in many pathogenic bacterial species, including the model organism and putative pathogen Escherichia coli, and may play a role in antibiotic resistance. Here we explore its physiological role during infection. Pathogenicity assays are being performed using the infection model Galleria mellonella, to study the phenotypes and survival rates following larvae infection with E. coli K12 and variants lacking rtc genes. Larvae infected with wild-type bacteria survive for up to 10 days as opposed to those lacking any of the rtc genes, which survive for up to 14 days on average. Complementation of the rtc gene deletions with wild-type Rtc proteins, but not functionally catalytic mutants, reverses the infection phenotype to that of wild-type. Similarly, viable larvae infected with wild-type or complemented bacteria score much lower in a health index scoring system than those infected with strains either lacking any of rtc genes or expressing catalytically inactive Rtc proteins. To further explore the importance of Rtc in infection, bacterial burden assays will be conducted to measure the bacterial load. The results so far suggest that bacteria with a fully functional Rtc system are more aggressive in the G. mellonellainfection model. This outcome supports the notion that the Rtc RNA repair system is involved in bacterial virulence and opens the window for further research to understand the ways in which Rtc may contribute to resistance, with potential for broader health benefit.

Conventional disease surveillance for shigellosis in developing country settingsrelies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.

Maedi-Visna is a lentivirus of sheep that causes lung disease and chronic wasting. It has been designated an “Iceberg disease” by the UK sheep industry levy board with a very large burden of subclinical disease that is often not apparent until losses in an individual flock become catastrophic. Disease prevalence in the UK is thought to have doubled in the last 10 years, however farmer and veterinary awareness of the disease is poor. There is no vaccine and treatment is not cost effective, meaning that the only realistic control option is culling of affected animals.

Current testing protocols use MV gag protein ELISAs. A long lag time between infection and antibody production means that many animals are missed on flock screening and repeated rounds of testing over a period of years are necessary to remove all infected animals. Preliminary testing of flocks that have attempted eradication indicates that those that do not keep testing until all animals are negative fail to eliminate the disease and that prevalence rates can even increase substantially in these flocks. The viruses extreme variability confounded attempts to develop a qPCR capable of detecting all variants, indeed deep sequencing was required to establish which strains of virus are currently present in UK sheep as there has been substantial genetic drift since the last sequencing studies (performed more than 20 years ago). More promisingly virus was detectable in nasal swabs of experimental animals at least offering sampling methods that can be done by farmers themselves.

The current study aimed to isolate and characterize bacteriophage against drug resistant, Shiga toxigenic E.coli, one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n=35) from neonatal calves, dairy workers and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multi-drug resistant to important antibiotics like as ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin and tetracycline. Bacteriophage namely Ib_pec2 was isolated against NM—18-040 isolate and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophage belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. SDS-PAGE analysis of structural proteins followed by LC-MS/MS analysis could able to identify five proteins identical to Tequatrovirus of myoviridae family. One step growth curve experiments revealed a latency period of 40 mins and a burst size of 893 PFU/ bacteria. Temperature and pH ranging from 40-50°C, pH 7-8, respectively were ideal for phage survival and multiplication. Phage was able to lyse 22 out of 35 STEC isolates. Thus, the study could able to characterize Ib_pec2 which could be used in control of STEC in the near future. STEC is a commensal organism in the gastrointestinal tract of ruminants but pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control its growth in ruminants and prevent its further spillage in the environment.

C. difficile, an anaerobic spore-forming intestinal pathogen, produces up to three toxins that cause host cell damage resulting in disease, C. difficileinfection (CDI). Therapies include antibiotic treatment; however, up to 30% of cases fail primary therapy, resulting in recurrent disease, which increases patient morbidity and places a burden on worldwide healthcare systems. We have little understanding of why these therapies fail. Using a clinically validated in vitro gut model, we assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. During induction of simulated CDI, C. difficile spores and vegetative cells became associated with the colonic biofilm microbiota. Vancomycin treatment did not effectively remove the biofilm C. difficile cells and recurrent infection was observed. Additionally, vancomycin therapy followed by faecal microbiota transplant did resolve the recurrent infection, but the biofilm C. difficile cells remained unaffected. In a biofilm transfer experiment, we showed that transferring biofilm encased C. difficile cells into a C. difficile naïve (but CDI susceptible model) induced CDI. Furthermore, we show that members of the biofilm community can impact C. difficile biofilm formation either acting in an antagonistic or synergistic manner. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.

Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls.

PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests.

Streptomyces coelicoloris a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosisas an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Germination assays show that spores of Δmcegerminate earlier than WT S. coelicolor, and impression mounts indicate spore chains emerge earlier in the Δmcestrain. As spores of Δmce possess an altered spore coat, it was hypothesised they might show reduced resistance to stressors. Heat kill assays show that deletion of the mce operon results in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. Spores of the Δmcestrain are also less resistant to triclosan, and sulfobetaines. Preliminary results indicate that the mce operon of S. coelicolor may be a cholesterol importer, as Δmce shows reduced growth in the presence of cholesterol, compared to WT S. coelicolor.

Although commonly thought to be only a measure of academic performance, assessments can also provide students with an opportunity to apply skills and knowledge acquired at university into written literacies that prepare them for the transition into prospective careers. Within a Level 2 Microbiology and Immunology course, students struggled to engage with standalone timetabled careers-related sessions, yet they showed enthusiasm when employability was embedded into assessments. A staff-student partnership project explored these issues, with the overall aim of understanding how to effectively embed employability skills into assessment and feedback, which supports the theme of supporting students to position themselves for the future.

Through student-run focus groups, this project investigated the reasons for low student engagement with the timetabled employability session and used student views to develop digital initiatives that could be applied to assessment and feedback practice with an emphasis on developing 21st-century competencies. These initiatives were then implemented as pre-session self-directed activities, which helped students link course feedback with employment skills and future career planning, followed by a newly developed in-class reflective feedback session that allowed students time to consider what skills they have developed and make links with future careers. Project evaluation was conducted using a quantitative survey of students involved.

This project aims to make feedback more meaningful and better valued by students. The approaches were designed in partnership with former students from the course, this project possesses the unique ability to deliver a genuine student voice both on employability concerns and the role of assessment.

Methylation by the type I restriction modification system (RMS) SpnIII in Streptococcus pneumoniae is hypothesised to regulate gene expression via epigenetic changes (Manos). The phase variable SpnIII generates six host specificity determinants (hsdS) alleles through site-specific recombination (DeSteCroix), each allele correspond with different methylation pattern.

In addition to known functions of the RMS in restricting phage infection (Furi) and transformation (Kwun), we have now tested impact on gene expression. RNAseq was used to analyse S. pneumoniae strains expressing a single spnIII allele (spnIIIA, spnIIIB or spnIIIE) to determine differences in gene expression profiles. The data have identified six genes which show differential expression and have a methylation site mapping to their predicted promoter region. Three synthetic promoters with the wild type and altered methylation target site were cloned in front of a luciferase gene in strains expressing a single spnIII alleles. For the first three sets of constructs analysed, data indicate that the methylated promoters show a three- to twenty-fold higher activity compared to non-methylated promoters. Results obtained were further confirmed by qPCR analysis. Preliminary data using drugs targeting DNA topoisomerases indicate that the mechanism by which methylation impact gene expression could be related to DNA topology. These data demonstrate for the first-time RMS dependent variations in gene expression and propose an alternative mechanism for this epigenetic regulation.

References:

* Manso, Nature Communication, 2014; 5:5055.

* De Ste Croix, J. Bacteriol. 2019; 201(15). pii: e00233-19.

* Furi, J. Bacteriol. 2019; 201(19). pii: e00370-19.

* Kwun, Nucleic Acids Res. 2018; 46(21):11438-11453.

Gonorrhea is a sexually transmitted disease caused by Neisseria gonorrhoeae and is one of the leading reportable STDs in adults, with ~87 million cases annually and globally. Gonorrhoea remains a major global public health concern not only because of rising incidence each year, but also because of rising antimicrobial resistance. There is an urgent need for long-term solutions to prevent gonorrhoea such as vaccines, but none currently existand research is focused on identifying potential antigens for inclusion in new vaccines. In our study, we used an immuno-proteomics approach to try and identify potential vaccine candidates.

A heterologous N. gonorrhoeae strain P9-17 was grown under iron-limiting conditions and whole cell lysates prepared. These were separated by isoelectric focusing (pH 3-10 range) and fractions were separated by SDS-PAGE. Western blots were prepared and reacted with sera from 20 patients with uncomplicated gonorrhoea and with sera from 5 controls with no history of gonorrhoea. Immuno-reactive bands were excised from the corresponding gels and subjected to mass spectrometry, which provided a profile of gonococcal proteins. After comparing patterns of reactivity, we identified 180 bands in sera from gonorrhoea patients. Using a bio-informatics approach we refined the list to 18 bands of interest and identified 13 novel proteins associated with an increase in immuno-reactivity with gonococci.

In summary, we have identified a unique set of gonococcal proteins that have not yet been investigated as potential vaccine antigens and that are the focus of current studies to develop a gonococcal vaccine.

Metabolism of pathogens in infectious diseases is important for their survival, virulence and pathogenesis. Mycobacterial pathogens successfully scavenge multiple host nutrient sources in the intracellular niche. It is therefore important to identify the intracellular nutrient sources and their metabolic fates in these pathogens. Metabolic phenotype of an organism is defined by metabolic fluxes. We quantified in vivo fluxes of the pathogens and probed host-bacterial metabolic cross talks in tuberculosis (TB) and leprosy using systems-based strategies and techniques of isotopic labelling, metabolic modelling and metabolic flux analysis (MFA). We show that the TB pathogen metabolizes a number of carbon and nitrogen sources in human macrophages and identified vulnerable nodes such as glutamine and serine biosynthesis as potential drug targets. Mycobacterium leprae, the leprosy causing pathogen, uses host cell glucose in infected schwann cells and the enzyme, phoenolpyruvate carboxylase is a potential drug target. Our research provides an understanding of the intracellular diets and metabolism of these important human pathogens and identified vulnerable metabolic nodes that can be used for developing innovative chemotherapies in TB and leprosy.

Mycobacterium tuberculosis is a leading cause of death worldwide and with increasing cases of drug-resistance, new treatments options are required. PrsA is an essential protein in mycobacteria and is necessary for arabinogalactan production and nucleotide biosynthesis. This protein is not currently targeted by any drugs in clinical development and represents a potential drug development avenue.

Currently, no crystal structure of M. tuberculosis PrsA (MtPrsA) is available, hence the SWISS-MODEL structure, based on M. smegmatis (93% similarity) was utilised. Molecular Dynamic simulations were performed on the modelled structure, to gain information on the protein’s predicted flexibility. The output trajectory was clustered, and representatives of each major cluster were used in downstream analysis. Ensemble docking, molecular docking on the ensemble of MtPrsA structures, of the GSK-177 prioritised compounds was undertaken. The docking predictions were also re-evaluated using machine learning based programmes, including NNScore.

This approach yielded several compounds which could be taken forward for further computational analysis. The compound rankings were also retroactively compared to experimental data studying the GSK-177 compounds’ interactions with mycobacteria.

This research represents a potential workflow for future in silico drug development efforts against M. tuberculosis and further work may allow the identified compounds to be used for the treatment of TB.

New enzymes and biocatalysis could provide routes to help limit global plastic pollution, as suggested by the example of PETases for the PET degradation. Experiments were conducted to screen soil microorganisms for enzymes able to break down the plastic polymer Lycra, which contains polyurethane linkages and could be utilized as a carbon and nitrogen source.

The methodology is based on a plate assay, which compared the growth of bacteria at 50°C and 65°C on minimal media and minimal media with a top layer, that contained DMF-dissolved Lycra. The medium provided the main nutrients required for bacterial growth, with Lycra being the only substantial carbon source. Promising candidates were identified as bacterial colonies with visibly enhanced growth on the Lycra-enriched minimal medium plate. The selected soil microorganisms were then tested with a liquid culture assay, where the strain was cultivated at 50°C or 65°C in liquid broth medium, that contained dissolved Lycra. The liquid culture samples were collected at 4 time points between 0-48h, and the aliquots tested on HPLC for detection of released Lycra monomers.

Around 50 different bacterial strains from 6 various soil and compost sources were tested with the described plate screening method and 7 bacterial thermophilic species were detected, that showed visibly enhanced growth on the Lycra-enriched plates. These results could support the development of Lycra enzymatic degradation. New enzymes could be engineered, optimized and yield a sustainable solution for the textile industry, which would not require toxic chemicals or contribute to landfill.

Production of therapeutic proteins is a $200 billion industry with E. coli being one of the main expression systems used and proteins often being secreted to the periplasm to produce disulphide bonds. However, little is known about how E. coliresponds to secretion of these therapeutic proteins into the periplasm under industrial conditions.

Proteomics and transcriptomics were conducted on cultures grown at high cell density in industrial production conditions to look at the effect of secreting a model therapeutic protein (scFv) to the periplasm compared to expressing the same protein in the cytoplasm.

82 proteins and 1653 transcripts were differentially expressed when overexpressing the scFv in different cellular compartments. ScFv secretion had a significant effect on expression of genes involved in the envelope stress response. However, while certain envelope stress pathways where upregulated upon secretion others were downregulated (including expression of some periplasmic chaperones). Scfv secretion also led to an increase in expression of genes involved in degradation of membrane proteins and the SEC secretion system, as well as iron transport, Outer membrane porins and flagella operons.

In future and current experiments, we are testing whether knocking out and overexpressing a selection of these genes improves secretion and folding of scFvs and other recombinant proteins.

Flavivirus non-structural protein 1 (NS1) is present at high levels in patient blood and has been previously implicated in increasing virus acquisition by mosquito vectors, which may facilitate increased transmission. Therefore, flaviviruses transmitted by Aedes aegypti mosquitoes were hypothesised to have similarities within NS1, and potentially other NS proteins, that may correlate with mosquito-borne (rather than tick-borne or no vector) transmission. Representative flavivirus amino acid sequences were downloaded from the NCBI Protein database, aligned using viprbrc.org and then manually analysed to identify residues correlating with arthropod vector tropism. All available sequences for individual clinically important flaviviruses were then analysed on viprbrc.org to determine sequence conservation within viral species for those residues correlating with vector tropism. Overall, we found five residues in NS1 that were highly correlated with transmission by mosquitoes, had functionally relevant differences in amino acid side chain properties between mosquito- and tick-borne viruses and had conservation within viral species. Additionally, a further nine residues in NS3 and four residues in NS4A were identified using the same methodology. Importantly, for NS1, for which more data is available regarding its role in influencing transmission, the residues we identified have not been implicated in increased transmission thus far. Hence, future work would aim to test the impact of mutating these NS1 residues to elucidate whether they modulate NS1 secretion into the blood stream, virus replication and/or transmission. Gaining an understanding of the molecular determinants underpinning vector tropism could be useful in the creation of transmission-incompetent vectors.

MX80 bentonite has been selected as the buffer and backfill in a proposed method of long-term deep geological storage of nuclear waste. Extensive studies have been carried out on the geomechanical properties of MX80; however, it is not clear what effect microbes will have on its ability to function as an effective barrier. Specifically, in the UK, as carbon steel waste canisters will contribute iron oxides and rust products to the immediate environment, iron-reducing bacteria are of interest. Iron-reducing bacteria can reduce structural or external Fe (III) to Fe (II) and some species are adapted to high temperatures and low water availability, in keeping with conditions within the waste repository. Indigenous iron-interacting bacteria have been identified in compacted MX80 and microbially-influenced iron-reduction was observed in groundwater salinity up to 0.45M NaCl. Experiments investigating gas production, and silica-solubilising abilities of this community were carried out. Further experiments in pressurised test cells investigated microbial activities at the clay / steel interface. Significant increases in hydrogen production were observed when microbes were present, and biogenically influenced changes in structure and appearance of MX80 were seen in all experiments. Additionally, silica release occurred, likely coupled to metal / microbe interactions. Corrosion products differed depending on microbial presence following incubation in test cells. Biogenic transformation of clay minerals through iron reduction or release of silica to groundwater could significantly impact the geomechanical properties of MX80, as indicated by observed changes in clay plasticity, and ultimately this could affect the behavior of the material as a barrier.