- Volume 1, Issue 1A, 2019

The accessory genome of Pseudomonas aeruginosa (PA), is frequently perceived insignificant compared to the core genome, however typically contains temperate bacteriophage (phage) genomes that effect the bacterial host. PA presence in chronically infected lungs correlates with loss of lung function particularly in cystic fibrosis (CF) and non-CF bronchiectasis (nCFBR). This study focuses on isolates from chronically infected nCFBR patients isolated over a decade, shedding light on how temperate phages change over the course of a chronic infection with antibiotic treatment. As temperate phages insert themselves into the bacterial genome they also have the ability to cause genetic diversity to their host’s genome, which drives evolution at an increased rate. By analysing the PA genomes isolated from the chronically infected lungs of patients over a period of 10 years. It was possible to predict the temperate phages in the genomes as well as induce the phages from the bacteria and sequence them. This then identifies which phages are rooted within the genome and which are inducible and therefore may transfer, granting horizontal gene transfer between strains in the lungs. The aim of this study is to ascertain whether by comparing the phages longitudinally and horizontally (when multiple strains were seen within a sample) it is possible to determine if these phage have a role in the PA infection within the lungs becoming chronic. This may also give an idea to why these PA infections are chronic and are so hard to clear from the lung, which is yet unknown.

Despite ∼60 % of human pathogens being of zoonotic origin, the diversity of viruses in non-human hosts remains understudied. To investigate the viral diversity present in Vietnam, the Vietnam Initiative on Zoonotic Infections (VIZIONS) collected 2100 rectal swabs and faecal samples from several non-human hosts (including pigs, rats and bats) as well enteric hospital patients and individuals with high animal contact. Samples underwent metagenomic sequencing (Illumina HiSeq), assembly (MetaSPAdes) and screening for viral sequences (Blastn and DIAMOND Blastp). The majority (87.5%) of viral sequences identified in humans shared high nucleotide identity (>95 %) to previously described viral species, with the exception of sequences assigned to Anelloviridae and Picobirnaviridae. Approximately half (46.5%) of viral sequences identified in non-human hosts shared between 60 to 80% nucleotide identity to its closest match in GenBank. For all hosts other from rats the family with the highest frequency of low identity hits was Picobirnaviridae. Sources of diversity at a viral family level were mostly host specific, with pigs having diverse astrovirus and smacovirus sequences, rats for rotavirus and adenovirus and bats for coronavirus and parvovirus. These results highlight the historic bias of sequencing viruses from human hosts and the need to shift focus to non-human hosts. Pig, bats and rats in Vietnam harbor a large diversity of potentially novel viruses that may be threats to animal and human health.

In order to streamline the study of the pathogenic yeast Candida parapsilosis, we developed a plasmid-based CRISPR-Cas9 system (pRIBO) for gene editing in this species. However, pRIBO was not feasible for large-scale generation of mutant strains. We recently addressed this bottleneck by generating pCP-tRNA, an improved version of pRIBO in which the guide sequence can be easily cloned into the SapI-digested plasmid, and the release of the mature sgRNA is mediated by endogenous RNase cleavage of the C. parapsilosis tRNAAla, and by self-cleavage of the HDV ribozyme. We are currently using pCP-tRNA for the systematic generation of mutant strains. Suitable guides were computationally designed to induce Cas9 cleavage within the first 25 % of each ORF in the genome, which is then repaired by recombination with a repair template (RT) containing 30 bp homology arms, 11 bp to introduce a stop codon in the functional reading frame, and a unique tag. In 4 months, 288 plasmids targeting genes encoding transcription factors, phosphokinases, or unknown functions, were transformed into C. parapsilosis with the corresponding RTs. The system resulted in gene editing of 62 % of the 288 genes at high efficiency (80–100 % of the colonies tested were positive); 16 % of the genes in the panel may be essential based on homology with related species, and we believe that the remaining 22 % may be successfully edited by selecting a different guide. In conclusion, we demonstrate that pCP-tRNA is a valuable tool for high throughput generation of mutants in C. parapsilosis.

Transcription Factor Decoys (TFDs) are short synthetic oligonucleotides that contain the binding site for specific bacterial transcription factors. When translocated into bacterial cytoplasm they can rapidly kill cells when targeted against essential bacterial pathways. Translocation is currently achieved by combination of the TFD with a proprietary lipidic delivery agent, CM2, to form nanoparticles. These interact with highly conserved anionic phospholipids, such as Cardiolipin, to effect delivery to both Gram-positive and Gram-negative bacteria. Simplifying translocation would be an advance that would allow serial screening of large libraries of TFDs to delineate genetic regulatory networks in numerous types of bacteria, including emerging strains. To achieve this we have combined key chemical moieties of the CM2 delivery molecule to the oligonucleotide conjugate by Click chemistry and show that these discrete conjugates are capable of translocation. Confocal laser scanning microscopy was used to monitor the uptake of the TFD-conjugates to E. coli and in parallel their effect on the targeted genetic pathways was confirmed with reporter strains and plating under selective conditions. Hence, it was confirmed that these conjugates can be used as tools to efficiently and specifically modify gene expression by inhibition of selected transcription factors.

Bacteria exist in complex communities that include not only many other bacterial species but also diverse mobile genetic elements which accelerate bacterial evolution via horizontal gene transfer. How multiple mobile genetic elements interact in bacterial populations and how this affects plasmid dynamics remains poorly understood. We experimentally evolved populations of Pseudomonas fluorescens SBW25 either singly-infected or co-infected with two conjugative mercury resistance plasmids either with or without positive selection (i.e. addition of Hg(II)). We show that co-infection led to higher levels of mercury resistance in the bacterial population in the absence of positive selection. Consistent with this, in the absence of positive selection the plasmids could stably coexist within bacterial cells resulting in the maintenance co-infection. By contrast, with positive selection, plasmid coexistence was destabilised, leading to the dominance of a single plasmid in several replicate bacterial populations. Plasmid co-infection appears to alter the trajectory of compensatory evolution to ameliorate the cost of plasmid carriage and may have selected for alternative mechanisms compared to singly-infected populations. Stable plasmid co-infection without positive selection for plasmid-encoded traits suggests that environments where plasmids are useless may be hot-spots for genomic innovation via plasmid-plasmid recombination.

Acetic acid has long been known for its antibacterial activity. We are using TraDIS to investigate the molecular mechanisms by which acetic acid acts as an antibacterial agent. To do this, we grew a high-density transposon library in uropathogenic E. coli EO499 serotype 131 in M9 media at pH 7 and pH 5.5 with acetic acid concentrations of 40 mM and 4 mM, respectively, or without added acetic acid. Sequencing libraries were generated from total bacterial populations after growth, and sequenced using a transposon-specific primer to generate positions and frequencies for each transposon. By comparing numbers of reads before and after the stress, we identified candidate genes where transposon inserts led to a decrease of fitness under acetic acid stress. Eight of these were chosen for further study: nuoM, nuoG, sucA, sthA, pitA, apaH, rssB and ytfP. Because of the difficulties of constructing gene deletions in the uropathogenic strain for validating the TraDIS results, we tested the relative fitness of the corresponding gene deletion mutants from the Keio library (in strain BW25113), with the growth conditions used for EO499. Interestingly, only a few knockouts showed a reduction in relative fitness in time course competitions at pH 5.5 with acetic acid. This may due to the differences between strains used in TraDIS and competition. To overcome this issue, we have also isolated transposon mutants from E. coli EO499 transposon library for the determination of relative fitness. The results will be presented.

C. parapsilosis causes candidiasis especially among newborn babies. The function of specific transporters is considered to be one key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS). Particularly, Some members of members of the drug/H (+) antiporter family (DHA1) of the MFS superfamily function as multidrug transporters. We find that the DHA1 family in Candida species can be divided into several clades. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (1 member in C. parapsilosis); TPO4, associated with polyamine transport (1 member in C. parapsilosis); NAG3/4, associated with transport of N-acetyl glucosamine (2 members in C. parapsilosis); TPO2/3, associated with polyamine transport (1 member in C. parapsilosis); YHR048w, with no known function (no members in C. parapsilosis); and TPO1/FLU1, possibly associated with fluconazole resistance (8 members in C. parapsilosis). We propose to use CRISPR-based gene editing to explore the function of all 13 members of the DHA1 family in C. parapsilosis. To date we have individually edited 10 members of the family by introducing stop codons near the start site of translation (ATG). We are currently editing the remaining 3 genes, and we are attempting to combine at least two edited genes in the same background. We will then test the phenotype of each edited strain in the presence of various drugs.

Enterohaemorrhagic E. coli O157 produces different Shiga toxin (Stx) subtypes which can contribute to the development of disease. The advancement of severe disease such as haemolytic uraemic syndrome is significantly associated to Stx2a subtype carriage. Three distinct EHEC lineages exist, where in the UK, stx2a has found in three STEC O157 sub-lineages: Ic, I/II and IIb. Stx encoding phages from the three sub-lineages where examined to determine whether the bacteriophage which conferred the stx2a synthesis are the same or different. Relevant representative EHEC O157 strains were sequenced using the MinION Platform. The sequences were assembled and annotated, allowing Stx encoding prophage identification. Such prophages and constituent genes were then aligned for comparison along with various reference strains. Results reveal that outbreak EHEC strains carry Stx2c encoding prophages and that such prophages were conserved, supporting past studies which suggest a single integration event and clonal expansion of Stx2c bacteriophages. Analysis of Stx genes revealed that some Stx2c phages carry Stx2a encoding genes, suggesting recombination between different Stx encoding phages. Variability was observed for Stx2a encoding phages, suggesting that there are various Stx2a encoding phages circulating in the UK.

Cancer has been identified as a group of diseases characterized by abnormal cell growth. In eukaryotic cells, the nucleolus is the region of the nucleus that constructs ribosomal subunits. Polymerase 1 transcription site has been used as the marker to particular aggressive tumours seen when the nucleoli increases in size and number. The nucleolar size correlates with the level of rRNA synthesis, which is transcribed by RNA polymerase 1 (RNAP1) during the initial stage of ribosome biogenesis. In recent years the rRNA transcription has emerged as novel target for anti-cancer therapy and specific RNAP1 transcription inhibitor are currently undergoing clinical trials for anti-cancer therapy. Thus, the proposed therapeutic strategies for solid tumour growth or inhibition of cancer cell proliferation is the selective inhibitor RNAP1 transcription. The currently available inhibitors are characterised by a different mechanism and different levels of genotoxicity. Two drugs, (CX-5461 and PMR-116) are small molecules and selective inhibitor of RNAP1 transcription which have moderate effect on transcription by other nucleolar polymerases and protein translation. CX-5461 inhibits transcription by displacing essential promoter recognition factor SL1 thus preventing an initiation of transcription. PMR-116 demonstrated a great potential as RNAP1 inhibitor, is characterized by low cytotoxity and very high anti-cancer effect. However, the exact molecular mechanisms of RNAP1 inhibition is unknown. Therefore, this experiment aims to identify the stage of the transcription cycle affected by PMR-116 by using a combination in vitro and vivo based assays. Also, to determine the drug target into the molecular mechanism of PMR-116 by using biochemical methods including Isothermal Titration Calorimetry (ITC).

Laboratory-based evolution has become a widely used method to explore fundamental questions about evolution as a process, and is also a powerful tool to study the link between genotype and phenotype. We have evolved six populations of E. coli MG1655 by iterative growth and dilution at pH 4.5 over five months, keeping a frozen fossil record of intermediate populations during this process. Whole genome sequencing of the evolved strains revealed many striking similarities in the evolutionary trajectories in the evolved strains; for example, mutations in arcA are common and may cause loss or alteration of function. We are interested in exploring the impact of different parameters on evolutionary trajectories, but as evolution experiments take a long time, we are currently investigating the potential of traDIS to replicate evolution experiments in a relatively short time frame. Since TraDIS provides a measure of relative contributions to fitness of each gene (by comparison of read counts after growth in two conditions), in principle it should be possible to use TraDIS to identify genes whose loss of function provides a fitness benefit. Here we compare the outcome of these two techniques and present our latest results.

The human gut microbiota includes viruses, termed the ‘virome’. Outnumbering the bacterial abundance on average 10 : 1. Recent studies suggested that changes in intestinal virome may lead to chronic gastrointestinal (GI) inflammation and bacterial dysbiosis. Thereby causing diseases such as inflammatory bowel disease (IBD), type I diabetes (T1D) and myalgic encephalomyelitis (ME), also known as chronic fatigue syndrome (CFS). Conversely, bacterial dysbiosis caused by increases in aerobic bacteria and decreases in anaerobic Bacteroides spp. have been described in some inflammatory mediated diseases and in considering that Bacteroides spp. are prominent members of the normal gut flora, their associated bacteriophages merit investigation. In this study we characterise Bacteroides related bacteriophages and their genomes isolated from sewage waste water environment. As part of this study we have made direct comparisons of Illumina HiSeq PCR-free and Oxford nanopore MinION PCR-free sequencing for viral metagenomics in addition to determining the extent of PCR related biases sequence generated viromes.

Biclustering algorithms are unsupervised machine learning algorithms that find paired subsets of samples and variables exhibiting co-dependence in a transcriptomics dataset. While matrix-factorization-based biclustering is especially suited to revealing enrichment patterns in metabolomic datasets, a full matrix-factorization-based biclustering pipeline does not exist. Here we present mfBiclust, an R package with a Shiny-based GUI that enables users to apply recently developed biclustering pipelines to the transcriptomics datasets. In our general matrix-factorization pipeline, a data matrix is approximated as the product of two factors. The optimal number of biclusters for a dataset can be estimated by bi-cross-validating truncated singular value decompositions. Biclustering results can be visualized and exported, facilitating functional characterization of the observed biclusters. mfBiclust is thus potentially useful for analyzing any genomics and transcriptomics assay.

Multidrug resistant (MDR) K. pneumoniae is listed among the most urgent public health threats due to its virulence and insusceptibility to a wide range of antimicrobials. Infection with this pathogen frequently leads to fatal outcomes, especially in low-income hospital settings. Patan Hospital is a 450-bed government hospital located within the Kathmandu Valley, Nepal. The hospital has previously witnessed multiple outbreaks caused by MDR K. pneumoniae in the neonatal intensive care unit (NICU). Particularly, a carbapenemase producing sequence type (ST) 15 K. pneumoniae clone was responsible for an outbreak with mortality rate up to 75 % in 2012. Recently in 2015, this same NICU suffered again from an MDR K. pneumoniae outbreak. In this study, using whole genome sequencing (WGS) and state-of-the-art analytic approaches, we aimed to define the nature of this recent outbreak. We found that the 2015 outbreak in Patan Hospital was caused by the same MDR ST15 K. pneumoniae reported in 2012. Albeit genetically similar, these recent strains were susceptible to carbapenems due to deletion of the blaNDM-1 cassette. Using Bayesian phylogenetic inference, we determined the outbreak strain was introduced to Patan Hospital in late 2010 and subsequently caused major outbreaks in NICU in 2012 and again in 2015. This clone acquired four different plasmids encoding resistance to numerous therapeutic antimicrobials, and this may underlie its successful propagation and associated high mortality. Insights provided through this study are invaluable in tailoring infection control strategies as well as raising public awareness

Lung tumors and infections remain the most common leading cause of mortality worldwide. Chaga mushroom (Inonotus obliquus) has long been considered the king of medicinal mushrooms which constitutes an inexhaustible source of active compounds which can affect the survival of tumor cells. It has been used widely for centuries as a dietary supplement and tea. In this sense, the aim of this study was to investigate in vitro cytotoxic capacity of water extracts of I. obliquus (Chaga mushroom) against the human lung cancer A549 cell line after 72 h incubation. In addition, the extracts were screened for antifungal activity on Aspergillus fumigatus species, a life-threatening cause of invasive pulmonary aspergillosis. The cytotoxic and the antimicrobial effects were performed using the MTT assay and the minimum inhibitory concentration (MIC) test, respectively. Owing to the noticeable effect on antiproliferation of hot-water extracts, especially those from I. obliquus, the extract could be of great potential to be used as an alternative cancer therapy. However, it was not proven to have antifungal effect against A. fumigatus fungi.

Nutritional quality and microbial density of sweet potato based complementary food fortified with soybean and crayfish flours was investigated. Three different samples were produced from mixing sweet potato, soybean and crayfish at different formulation. Sample I, II, III are sweet potato: soybean: crayfish at ratio 80 : 15 : 5, 70 : 20 : 10 and 60 : 25 : 15 respectively. The formulated flours were analysed for proximate composition and microbial density. The result of proximate composition showed that the value of moisture content ranged between 6.50–8.50 %, while ash content, crude protein crude fat, crude fibre, carbohydrate and energy value ranged from 5.50–9.50 %, 25.10–28.50 %, 4.20–6.20 %, 0.13–0.27 %, 47.73–56.27% and 363.42–370.48 Kcal/100 g respectively. Its observed that as the level of inclusion of soybean and crayfish to sweet potato flour increases, there was increase in protein and crude fibre content but decrease in carbohydrate content. Also, the total bacteria count, coliform count, yeast and mould count ranged from 28 to 50×10–4, 18–37 x 10–4 and 40–62×10–4 (c.f.u./g) respectively which are within the recommended limit value for microbial density in food. Therefore, sweet potato flour fortified with soybean and crayfish flour can be recommended as weaning food to reduce the incidence of malnutrition in infants.

Food contamination with aflatoxin is more prevalent in tropical regions where environmental conditions such as high temperature and humidity prevail, which favour the growth of toxigenic fungiand accumulation of these toxins in food and feeds represent a major threat to human and animal health. In lieu of the previously known adsorbents, adsorption studies of Aflatoxin (AF) were performed using inexpensive, readily available and local adsorbents Imarsil and activated charcoal (AC). Fifteen edible oils were purchases from open markets in Nigeria and screened for aflatoxin using High Performance Liquid Chromatography (HPLC). Thirteen out of the fifteen vegetable oil samples were positive to aflatoxin at the following concentration(172, 123, 195, 142, 46, 107, 96, 116, 22, 33, 228, 17 and 4) ng/kg while two had no detectable AF. Atsix different concentrations (0.5, 1, 1.5, 2, 2.5 and 3%) of Imarsil and activated charcoal (AC) with contact time of 1,2 and 3 h at room temperature (37 °C), the aflatoxin-adsorbing capabilities depend on the adsorbent concentrations and contact time. Imarsil demostrated 100 % adsorption efficiency within one hour. At AF contamination rates of 96–228 ng/kg, activated charcoal was not effective while Imarsil had 100 % removal efficiency within 3 h witha significant reduction (P<0.05) observed at the highest contamination rate and adsorbent concentration. AC demonstrated very mild adsorption activity. Results from this study indicated that Industrial incorporation of Imarsilinto the oil refining process would reduce greatly the menace of aflatoxicosis. Hence, the use of Imarsilshould be encouraged.

Technological properties of Bacillus spp. involved in fermenting Ceiba pentandra seeds into ‘kantong’ a condiment prepared and consumed in the northern part of Ghana were invested so as to develop starter cultures with the predominant species for commercial production of ‘kantong’. The Bacillus species which predominated 205 Bacillus strains isolated from 11 stages of ‘kantong’ production includes: Bacillus amyloliquefaciens, B. safensis, B. altitudinis, B. thuringiensis, B. pumilus, B. megaterium, B. cereus, B. circulans, B. coagulans, B. firmus, B. subtilis and B. licheniformis. These predominant species were assessed for some technological properties such as proliferation at different temperatures and pH; substrate utilization preferences including ‘kantong’ formulated media (i.e. 48 h sample, dried pellet and the final product) and inhibitory activity against 12 selected pathogenic and spoilage microorganisms. The strains proliferated at different temperatures between 10 °C and 55 °C and pH of 2 to 9. Substrate utilization preferences were Nutrient Agar with 5–9 % Sodium Chloride (NA/NaCl C, and ordinary Nutrient Broth [NB], Nutrient Agar (NA), Potato Dextrose Agar (PDA), Tryptone Soya Agar (TSA), MacConkey Agar (MCA) and ‘kantong’ formulated media. All strains exhibited inhibitory activity against one or more pathogenic and spoilage organisms. Salmonella typhimurium, Staphylococcus aureus, E. faecium and Proteus vulgaris were the most susceptible indicator microorganisms. Many strains qualified as potential candidates for selection and development as starter cultures to be used in the large scale commercial production of ‘kantong’ of consistent and acceptable organoleptic quality.

Finger millet in northern Nigeria was subjected to phytochemical screening using standard procedures. The agar well method was used to test the antibacterial activities of methanolic and aqeous (combined) extracts of the grain on Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The result of the antimicrobial activity as indicated by zone of inhibition ranged from 1 to 8 mm for different extract concentrations. The finger millet extract showed zones of inhibition of 8 mm against Pseudomonas aeruginosa at a concentration of 100 mg ml−1, 3 mm at 50 mg ml−1 and 2 mm at 25 mg ml−1 concentrations. The inhibition zones of Escherichia coli at extract concentrations of 100 mg ml−1, 50 mg ml−1, 25 mg ml−1, 12.25 mg ml−1 and 6.125 mg ml−1 were 4 mm, 3 mm, 3 mm, 6 mm and 1 mm respectively, and for Staphylococcus aureus were 5 mm,2mm, 1 mm at 100 mg ml−1, 50 mg ml−1 and 12.25 mg ml−1 respectively. The zones of inhibition against all the tested isolates at 100 mg ml−1 was not significantly different from those of 50 mg ml−1 (P=0.160), 25 mg ml−1 (P=0.067) and 12.5 mg ml−1 (P=0.160), but significantly higher than 6.125 mg ml−1 (P=0.05). Although S. aureus and S. typhi also did not differ significantly in their susceptibility to the varying concentrations of the extract (P=0.157), but susceptibility by S. typhi was significantly lower than those of E. coli (P=0.007) and P. aeruginosa (P=0.015). The qualitative phytochemical analysis indicated the presence tannin/phenol, flavonoids, alkaloid, saponin, glycosides, terpenoid and steroids in finger millet. The quantitative phytochemical revealed total phenolic content (6.57 mg/100 g) and total flavonoid content (0.224 mg/100 g). The overall results indicate that finger millet are potent antimicrobial preparations at least in vitro and also have high nutritional value.