- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Genetics and Genomics Forum
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Dissemination of a phage-encoded virulence factor in a pandemic S. Typhimurium
More LessSalmonella Typhimurium is the second most common cause of foodborne salmonellosis. Phage-mediated horizontal gene transfer contributes to the virulence of S. Typhimurium. An example of a phage-encoded virulence gene is sopE, a T3SS effector, found rarely in Typhimurium and associated with epidemics. The current pandemic and multi-drug resistant monophasic variant of S. Typhimurium (S. 4,[5],12:i:-) acquired sopE in multiple events following the lysogeny of a previously undescribed bacteriophage, mTmV. The current study aimed to further investigate the association of the virulence gene with S. 4,[5],12:i:- and assess its epidemiological impact. To this end, a large collection of clinical S. Typhimurium isolates from the UK have been analysed for the sopE gene and mTmV presence using a phylogenomic approach. While a large proportion of S. 4,[5],12:i:- (41 %) carried the sopE gene, few isolates outside the epidemic clade harboured it. Notably, the mTmV bacteriophage was identified only in S. 4,[5],12:i:-, although laboratory experiments demonstrated that the phage host range is not restricted to it. Nonetheless, we identified the phage in other S. enterica serovars circulating in the same ecological niche of S. 4,[5],12:i:-. In addition, a genomic characterisation of mTmV was performed revealing an unexpected level of phage variation. Finally, we identified a novel phage-like element harbouring the gene. The study revealed the large dissemination and selection of the virulence gene in the current epidemic, which is mobilised by multiple and distinct mobile genetic elements.
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Comparison of temperate bacteriophages of Pseudomonas aeruginosa from the lungs of chronically infected non-cystic fibrosis bronchiectasis patients over a period 10 years
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.
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Investigating virus diversity in humans and non-human animals in Vietnam
More LessDespite ∼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.
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Large-scale generation of mutant strains in Candida parapsilosis using CRISPR-Cas9
More LessIn 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.
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Oligonucleotide transcription factor decoys as tools to control bacterial transcription
More LessTranscription 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.
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Do uropathogenic E. coli require changes before it can spread into the bloodstream?
More LessBackgroundUropathogenic Eschericha coli are the leading cause of urinary tract infections (UTIs). The microbe can spread from bladder to kidney and finally to blood. We wished to determine whether genetic changes accompany the passage of these infections from urine to blood.
Material/methods12 paired urine and blood samples were collected from patients in Greater Glasgow and Clyde; the interval between sample collection time between pairs was less than 48 h. Whole genomic sequencing of these paired samples was performed using the Illumina MiSeq platform. De novoassembly of reads was carried out using Shovill assembler; whereas SNPs were identified using SMALT, VarScan and Gubbins tools.
ResultsUrine and blood samples in each pair had the same MLST type. Surprisingly, however, there were multiple differences in the presence of plasmid genes, phage elements and insertion sequences within pairs, as well as numerous SNPs. For example, the mercury reductase merAgene and mercuric transport protein merPhave been acquired in a plasmid of a blood isolate compared to the contemporaneous urine sample. We also identified missense mutations in genes involved in several metabolic pathways in bloodstream isolates. Several of the observed gene deletions/insertions and SNPs were found in more than one of the paired blood and urine isolates.
ConclusionsThe observed sequence differences between contemporaneous blood and urine isolates suggests that genomic differences accumulate within the urinary bladder prior to blood stream invasion. The observed blood stream variants may thus possess a selective advantage in invasion and/or survival within blood.
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Coinfection promotes plasmid stability
More LessBacteria 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.
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Molecular approaches to understand the effect of acetic acid in uropathogenic E. coli
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.
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Investigation on MFS family in C. parapsilosis with CRISPR
More LessC. 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.
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Shiga toxin prophage analysis of clinically relevant enterohaemorrhagic E. coli isolates using Nanopore sequencing
More LessEnterohaemorrhagic 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.
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Investigation of molecular mechanisms of polymerase I (Pol-l) inhibitor PMR-116 using Isothermal Titration Calorimetry (ITC)
More LessCancer 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).
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High throughput approaches to study laboratory-based evolution of E. coli for enhanced growth at low pH
More LessLaboratory-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.
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Characterising the gut virome by cross comparison of sequence platforms and isolating bacteroides bacteriophages from sewage water
More LessThe 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.
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mfBiclust: an r package for biological bicluster analysis in the transcriptomics dataset
Quan Gu and Jonathan LimBiclustering 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.
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Genomic analysis reveals the re-emergence of a nosocomial outbreak caused by multidrug resistant Klebsiella pneumoniae
More LessMultidrug 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
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- Global Food Security: The Challenges for Microbiology
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Chaga mushroom (inonotus obliquus) inhibits growth of both lung adenocarcinoma (A549) cells and Aspergillus fumigatus
More LessLung 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.
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Nutritional quality and microbial density of sweet potato flour fortified with soybean and crayfish flours
More LessNutritional 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.
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Detoxifying potentials of two indigenous adsorbents: imarsil and activated charcoal in the reduction of aflatoxin in vegetable oils consumed in Nigeria
More LessFood 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.
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Starter culture development using selected strains of Bacillus spp. associated with “kantong” production in Ghana
More LessTechnological 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.
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Phytochemical and antibacterial property of finger millet (Eleusine coracana) on some selected clinical bacteria
More LessFinger 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.
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Carbapenemase-producing Enterobacteriaceae (CPE) isolated from pigs in China
More LessBackgroundThe increasing prevalence of CPE is a global concern in public health. CPE were found to be present in livestock. Food animals act as a reservoir for NDM-producing bacteria. However, there is limited information about CPE in food animals. Here we screen the carbapenemase-producing bacteria in pig samples.
ObjectiveTo investigate the prevalence of carbapenem resistance in swine from China.
MethodsA total of 138 rectal swabs from pigs imported from China were collected in Hong Kong between June 2017 and Oct 2018. Bacterial identification was conducted by MALDI-TOF for all isolates. Carba-NP test and disc diffusion method were performed to detect carbapenemase and determine the antibiotic susceptibility. Identification of carbapenemase gene and replicon type of plasmid, and further characterization of isolates were performed through PCR and next-generation sequencing respectively.
ResultsTwenty-one CPE isolates including Escherichia coli (n=20) and Enterobacter cloacae (n=1) were isolated from 20 pigs, which were resistant to carbapenem (meropenem, ertapenem and imipenem). The prevalence rate of carbapenemase producers was 14 % (20/138). All isolates were positive in carba-NP test and harboured carbapenemase gene bla NDM. Two-third of IncX3 (14/21) plasmid appeared in bla NDM-producing isolates. Different resistance patterns were discovered among NDM-carrying isolates, but all of them were susceptible to fosfomycin and azithromycin.
ConclusionOur data show that the prevalence of carbapenem-resistance Enterobacteriaceae among swine in China during 2017 to 2018 . It is also observed that NDM carbapenemases is still circulating in pigs over times.
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The use of microbiological methods to reduce aflatoxin M1 in cheese
Studies have shown evidence of human exposure to aflatoxin M1 due to the consumption of contaminated milk and dairy products (mainly cheeses). This poses a great risk to public health, since milk and milk products are frequently consumed by a portion of the population considered immunosuppressed, children and the elderly. Knowledge of the negative impacts of aflatoxins on health and economics has led to investigations of strategies to prevent their formation in food, as well as to eliminate, inactivate or reduce the bioavailability of these toxins in contaminated products This study evaluated the effect of microbiological methods using lactic acid bacteria on aflatoxin M1 (AFM1) reduction in Minas Frescal cheese (typical Brazilian product, being among the most consumed cheeses in Brazil) spiked with 1 µg l−1 AFM1. Inactivated lactic acid bacteria (0, 5%, v/v de L. rhamnosus e L. lactis) were added during the cheese production process. Nine cheeses were produced, divided into three treatments: negative controls (without AFM1 or lactic acid bacteria), positive controls (AFM1 only), and lactic acid bacteria+AFM1. Samples of cheese were collected on days 2, 10, 20 and 30 after the date of production and submitted to composition analyses and determination of AFM1 by high performance liquid chromatography. The reductions of AFM1 in cheese by lactic acid bacteria at the end of the trial indicate a potential application of inactivated lactic acid bacteria in reducing the bioavailability of AFM1 in Minas frescal cheese without physical-chemical and microbiological modifications during the 30 day experimental period.
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Integrated phenotypic and genomics analysis to elucidate differences in stress resistance and virulence of Listeria monocytogenes strains
Listeriosis is an important food-borne disease responsible for high rates of morbidity and mortality. L. monocytogenes has been the cause of several food-borne outbreaks and product recalls throughout the world. It can adapt and survive in a wide range of stress conditions which makes it difficult for food producers to eradicate. The goal of this study was to use phenotypic assays and whole genome sequencing to elucidate possible links between food related stress resistance and virulence phenotypes in L. monocytogenes strains originating from different sources. Four L. monocytogenes isolates from sweetcorn and one isolate from a food processing environment (control) were sequenced and evaluated for the ability to survive in acid (pH 3.5, 15 min), in the presence of a commercial antimicrobial mixture (2 % v/v, 90 min), heat (60 °C, 5 min) and hydrogen peroxide (420 mM, 15 min). Results showed that the strains had different resistance levels to the above stressors with the environmental strain being more susceptible to heat and the commercial antimicrobial. Also, results showed that the four sweetcorn isolates were more virulent than the environmental isolate as they had significantly higher attachment and invasion capacity onto HCT-8 cells (P Pan-genome analysis revealed that the four isolates fall within a class associated with recent outbreak strains. Single Nucleotide Polymorphisms (SNPs) analysis was performed on the five genome sequences and subsequent cluster analyses on the resulting whole genome SNP matrix revealed differences between the strains.
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Distance-decay patterns overshadow effects of long-term fertilization and tillage on microbial community structure in agricultural soils
More LessCommunity profiling is one of the most utilised tools in microbial ecology today. The relationships between microbial communities and their environment affect ecosystem function in fields spanning from medicine to agriculture; and understanding the community dynamics of a microbial community is key to understanding the complex effects of human intervention. In this study, we look at the effects of long-term tillage and fertilization regimes in soils from an agricultural block-designed field trial set up in 2001. By studying the microbial community composition, absolute microbial abundance and diversity of denitrification functional genes in the context of environmental data, we were able to address the question of how specific land management histories affect the diversity and distribution of bacteria and denitrification genes within agricultural soils. It was found that microbial communities appear to be largely unaffected by land management history, and cluster predominantly by spatial location within the field, despite lack of significant environmental variation. In this well-established agricultural field trial, Euclidean distance is the major identifiable determinant of microbial community dissimilarity (as well as dissimilarity in microbial abundance). That ecological drift, rather than physicochemical factors can be the major determinant of genetic potential may have consequences for attempts to understand nutrient availability in agricultural systems. Additionally, the overwhelming variation caused by spatial distance indicates that block designed experiments may not always have sufficient statistical power to identify any effects of human treatment.
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Incorporation of V. vulnificus into marine snow for oyster uptake and in vivo bacterial competition assays
More LessVibrio vulnificus is a significant human pathogen found in high numbers in oysters. Despite the environmental prevalence of V. vulnificus, clinical cases are uncommon. We hypothesised that in vivo competition between V. vulnificus and other strains/species resulted in the killing of hypervirulent strains, reducing clinical incidence. To assess this, we have developed an oyster model into which we can ensure ingestion of high quantities of V. vulnificus using a defined ‘marine snow’. Marine snow describes the aggregation of naturally occurring phytoplankton, bacteria, debris and other organic materials. Our marine snow substrate is comprised solely of the diatom, Thalassiosira pseudonana. Bottles containing artificial seawater, 109 T. pseudonana, V. vulnificus culture and hyaluronic acid were rotated at 16 r.p.m. for 24 h to generate aggregates. V. vulnificus-containing marine snow was added to beakers holding individual oysters. Following 24 h uptake, oyster stomachs were excised and homogenised in PBS. The resulting suspension was serially diluted for plate-counts and DNA extracted for downstream qPCR analysis. Diatom-based aggregates present a controllable and reproducible model for incorporating V. vulnificus into marine snow. Using this methodology, we demonstrate greater uptake of V. vulnificus by oysters than any current study. This has potential applications for future in vivo work studying a range of microorganisms in oysters, such as other human pathogens or those of interest to aquaculture. Future work will undertake in vivobacterial competition assays to determine the role that intra-/inter-species competition has on the ecology of V. vulnificus and whether this impacts the clinical incidence.
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Investigating the effect of alkaline stress on biofilm formation by Salmonella enteritidis
More LessBacterial biofilm formation is an important survival strategy in multiple environments. It is affected by the attachment surface, the bacterial strain and the surrounding environment. In Salmonella enteritidis, a biofilm-forming foodborne pathogen, the molecular biofilm regulators act as on-off controls between the sessile and the planktonic population, while the exact underlying formation mechanism still remains unclear. The aim of this project is to study the effect of alkaline environment on the formation of Salmonella biofilms and to examine the architecture of biofilm produced under alkaline conditions, by use confocal microscopy. Neutral pH was found to be the optimal pH for Salmonella biofilm formation, while pH 10 significantly reduces it (P-value=0.015). However, cell viability remains high at pH 10, which suggests that the pathogen can easily survive the alkaline stress. Biofilm morphology at pH 7 is characterized by thick cell clusters, whereas at pH 10 it is characterised by thin layers of individual cells. These findings can help us understand how Salmonella enteritidis survives under highly alkaline conditions, potentially leading to the design of new and more effective disinfection strategies involving highly alkaline detergents.
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Food inspection of the maltese cheeselet using hyperspectral imaging. The ‘food inspection using hyperspectral imaging’ (FIHI) project is financed by the Malta Council for Science and Technology, for and on behalf of the Foundation for Science and Technology, through the FUSION: R&I technology Development Programme
More LessThe island of Malta has a rich heritage, which is evident in the rustic appeal of this island. Ġbejna forms an integral part of the Maltese food heritage. This artisan product is made from sheep or goat milk curds and aged for several months to develop its distinctive taste. During the ageing process, the cheese can become spoiled by fungi and unsafe for human consumption. This is a significant public health risk and a financial liability for producers. Conventional microbiology techniques do not detect these slow-growing pigment-less fungi early, allowing occasional distribution of contaminated products. We propose the use of hyperspectral imaging to detect these fungi during the early stages of cheese production. In contrast with a typical digital camera, which compiles the light signal into three broad wavelength bands; red, green and blue, a hyperspectral camera records numerous narrow and contiguous wavelength bands reflected from an object. This produces a series of images, each corresponding to the reflected electromagnetic energy in the respective narrow band of wavelengths. This image series may, in turn, be used for early fungal detection and identification. To test this hypothesis, a model cheeselet was produced to conduct compatibility and stability studies, through measurements of colony forming units, water activity, moisture levels, pH, protein and sugar content. The ġbejna model was then challenged with fungal strains isolated from commercial ġbejna and imaged using a hyperspectral camera. An algorithm is under development to differentiate contaminated samples from uncontaminated samples using image analysis and multivariate statistics.
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Microbiome and resistome of the gastrointestinal tract of broiler chickens
More LessAntibiotics are used extensively in agriculture as therapeutics, and in some countries, for prophylaxis and as growth promotors. Alarmingly, there is a potential resistance transmission pathway from animals to humans through food. We analysed the microbiome and resistome of sixteen broiler chickens, which are raised for meat production. DNA was extracted from caecal samples taken at days 27 and 34 posthatch from broilers, half of which had their diets supplemented with a mannan rich fraction. Paired end sequencing was performed using an Illumina HiSeq 4000. The data was analysed by MGnify to generate taxonomic read files. The microbiome was analysed using Calypso software and the antibiotic resistance genes (ARGs) identified using the ARGs-OAP pipeline. The main phyla detected in all samples was Firmicutes and Bacteroidetes. Clostridia was the main class detected and Clostridiales the main order. Faecalibacterium, Lactobacillus and Bacteroides were the main genus detected, which are common in the broiler caecal microbiome. There was an increase in Bifidobacterium in the treated group. Principle component analysis showed that both time points cluster together. Rarefaction analysis confirms that a sufficient sequencing depth was obtained. Tetracycline resistance comprised the greatest proportions of ARGs present, followed by the aminoglycoside, macrolide, vancomycin, beta-lactam and bacitracin classes. Further analysis of the sequences will allow for full characterisation of the resistome between treatment groups. The antibiotic resistance residues in food animals may have the potential to disseminate antibiotic resistance to the human community via the food chain.
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Surveillance of Shiga-toxin producing Escherichia coli in Irish sheep
More LessShiga-toxin producing Escherichia coli (STEC) is a foodborne zoonotic pathogen of significant public health concern. Ruminant animals are considered the primary reservoir of STEC. STEC predominantly colonises the lower gastro-intestinal tract, termed the recto-anal junction (RAJ). The number of STEC shed in the faeces of ruminants can vary widely with some animals, termed ‘super-shedders’ (>Log104 c.f.u. g−1 faeces), high risk carriers of the pathogen. The objective of this study was to sample a large cohort of Irish sheep, with quantitative and qualitative analysis of each sample for STEC. RAJ swab samples (N=410) were collected over a 9 month period from an ovine slaughtering facility. Each swab was enriched in 30 ml of modified Tryptone Soya Broth with Novobiocin at 41.5 °C for 5 h and subjected to a quantitative real-time PCR assay to detect and enumerate serogroups O157 and O26 in super-shedding animals. Incubation was allowed to continue for 24 h and shiga-toxin prevalence was assessed using a targeted qualitative real-time PCR assay. Eight O157 strains were isolated, of which six were super-shedding strains. The incidence of stx, O157 and O26 positive swabs was 49.3 %, 1.95 % and 0.24 % respectively. The prevalence of stx1, stx2 and stx1/stx2 virulence factors in isolated strains was 15.9 %, 8.8 % and 22.4 %. Additionally, the occurrence of stx1/stx2 in combination with eaeA in strains was found to be significant according to Pearson’s correlation and a paired T-test. In conclusion, these results underline the risk Irish sheep pose as a potential source of STEC infection.
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Analysis of phenotypic traits which may impact long term survival of different Escherichia coli pathotypes
More LessShiga toxin producing E. coli (STEC) is a foodborne pathogen which causes severe, debilitating, and sometimes fatal, illness. Ireland consistently has one of the highest incidence rates of human STEC infection in Europe. Cattle are one of the primary reservoirs for STEC, excreting the pathogen in their faeces. The amount of pathogen excreted varies greatly, with animals shedding >log104 c.f.u. g−1 faeces being termed ‘super-shedders’. Human infection can occur from faecal contamination of meat, dairy, fresh produce or drinking water. STEC can survive for extended periods in soil, slurry and water, although the exact means is unknown. The objective of this study was to examine phenotypic traits potentially relevant to extended environmental survival in two strain banks: (1) clinical and bovine STEC, in comparison with non-STEC, isolated from the production environment and (2) E. coli O157:H7 of known shedding status. The strain banks were assessed for biofilm-forming abilities and the ability to adhere to the muscle component collagen-I, using a 96-well crystal violet assay, where the absorbance of bound cells indicated biofilm formation levels or adherence to collagen. Extracellular components involved in attachment were assessed using Congo red agar and pellicle formation was also examined. Phenotypic traits potentially related to extended environmental persistence were observed more frequently in non-STEC. However, these traits were also observed in some STEC isolates, showing phenotype is strain dependent indicating a risk for enhanced environmental survival of some STEC isolates. The shedding status of E. coli O157:H7 is not dictated by the investigated characteristics alone.
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Perceptions towards antimicrobial use and resistance in the UK pork supply chain
More LessAntimicrobial Resistance (AMR) occurs when micro-organisms develop the ability to counteract antimicrobial drugs through previous exposure. The past decade has seen an increased prevalence of AMR bacteria due to widespread antimicrobial use (AMU). A substantial share of antimicrobial consumption is attributed to animal production, particularly within the pig industry as it is recognised to be a high user of antimicrobials. Considerable research has focused on the scientific mechanisms of AMR; however, limited literature exists regarding the perceptions of food producers towards AMR. Four databases; Web of Science, PubMed, ScienceDirect and Google were used to search keywords; ‘UK,’ ‘pork,’ ‘supply’ and ‘chain’ to identify relevant papers. Each paper was inspected to ensure that a pork supply chain was illustrated. Interviews were conducted respectively with professionals working in the pork sector to verify the chain and uncover perceptions towards AMU and AMR. Results verified the accurate mapping of the pork chain, enabling professionals to highlight areas of AMU. Stakeholders perceived antimicrobials as useful for the treatment of diseases however, opinions varied regarding the transfer of AMR to humans and the effects this may have on health. To combat the problem of AMR and high use of antimicrobials within the pig industry, it is necessary to identify the key stages of AMU and to uncover stakeholder perceptions along the pork chain. Results will be verified using surveys and an intervention will be designed to enhance knowledge and understanding of AMR to influence a change in behaviour and thus, positively impact farmers on-farm practices.
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Proteomic analysis of three ubiquitous phytophthora species threatening global forest ecosystems
More LessPhytophthora are a genus of microbial, filamentous eukaryotes that morphologically resemble fungi but belong to the Oomycete class. Phytophthora species include some of the most destructive pathogens of plants, including many economically important crops and forest species. They represent one of the biggest threats to worldwide food security and natural ecosystems. Phytophthora are notorious for secreting large arsenals of effector proteins which facilitate infection by degrading host cell components, exploiting host nutrients, dampening host immune responses and inducing necrosis. Compared to other taxonomic groups, there is a paucity of OMICs data available to study Phytophthora species. To this end, we have used an LC-MS/MS strategy to perform the first large-scale profiling of the secretomes of three Phytophthora species that are an increasing threat to global forest ecosystems: Ph. chlamydospora, Ph. gonapodyides and Ph. pseudosyringae. Together, Ph. gonapodyides and Ph. chlamydospor are present the two most widespread Phytophthora species, having been found in a wide range of habitats globally. Ph. pseudosyringae has been identified as the cause of oak and beech decline across Europe and America. Here, we use mass spectrometry to characterise the secretome of these Phytophthora species by identifying proteins secreted into different growth media. We detect a number of important effector families including proteins involved in the breakdown of plant cell wall carbohydrates (CAZymes) and toxin families such as necrosis-inducing proteins. Our results provide important insights into understanding the molecular mechanisms of Phytophthora infection.
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Biotechnological approach to produce riboflavin enriched iru – using riboflavin overproducing Bacillus subtilis
More LessDietary deficiencies are major cause of malnutrition in the developing world, particularly vitamins deficiencies such as riboflavin, which lead to various health disorders. Traditional plant fermentation and their indigenous starter cultures such as Bacillus subtilis might provide solutions to bioenrich riboflavin. We aimed to investigate this idea on the example of B. subtilis derived from iru an alkaline fermented condiment playing an important role in rural Nigeria. After initial isolation, identification and safety assessment, roseoflavin exposure was used to obtain riboflavin overproducing mutants. These were further analysed for functional characteristics. Bàcillus species’ (n- 123) were isolated from iru. Initial riboflavin production in supportive growth medium ranged from 50.3 to 479.0 µg l−1 for 27 out of 123 strains evaluated. Subsequent gràdual exposure to 200 mg l−1 roseoflavin increased riboflavin production in the three best producing strains from 350 µg l−1 to 542 µg l−1, 479 µg l−1 to 580 µg l−1 and 362 µg l−1 to 618 µg l−1. This increased riboflavin in lab-scàle iru fermentation by over 150 percent to 0.12–0.14 mg g−1 and near the recommended daily intake while retaining desired proteolytic and esterase activity. This research provides important proof of concept for the bioenrich the of traditional B. subtilis based plant fermentations used across sub-Saharan Africa and possibly other areas globally.
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Commercial potential of plant growth promoting rhizobacteria on Amaranthus hybridus in Ede, Osun State
More LessThe eco- friendly improvement of crop yield is a mammoth task that must be tackled in order to meet the ever increasing world population in need of food. Plant growth promoting rhizobacteria (PGPR) are organisms known to increase the growth of plants, by directly or indirectly facilitating crop yield by a number of mechanisms. Vegetables are stable foods rich in numerous vitamins. Low income countries get a balance diet through regular consumption of vegetables like Amaranthus hybridus which is regularly consumed with highly starchy food across the Southern states of Nigeria. This study investigated the effect of different bacterial suspension samples in increasing the growth parameters of Amaranthus hybridus through different plant growth conditions and treatments, determination of rhizospheric and endophytic bacterial colonization, RNA extraction, cDNA synthesis and qRT – PCR analyses, and Microarray hybridization. Bacterization by microorganisms tagged ADK 1, ADK 2, ADK3, ADK4, ADK 5, ADK 6 and ADK 7. The pot trial showed an increase in the growth yield of Plant affected by ADK 5 and ADK 7. A synergistic effect of ADK 5 and ADK 7 did not give an increased yield as each individual effect. An Amaranthus hybridus transcriptome analysis revealed that several genes showed differential expression after inoculated by ADK 7. These genes are implicated in stress response and hormone pathways. Investigations into the bacterization of indigenous vegetables by indigenously isolated bacteria is an eco-friendly agricultural practice to be promoted.
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Detection of SHV, CTX-M and TEM genes in extended spectrum beta lactamase producing multi-drug resistant Escherichia coli from clinical isolates in Calabar, Nigeria
More LessBackgroundThe emergence of multi-drug resistant strains of Escherichia coli complicates the treatment of infections. The detection of ESBL genes in bacteria and their antimicrobial resistance patterns can provide information about their epidemiology and transmission. The study aimed to detect ESBL genes that encode; CTX-M, TEM and SHV in E. coli isolatesin our locality.
MethodsClinical E. coli isolates were obtained from public and private clinics within Calabar metropolis. Biochemical method was used to re-identify the isolates. Antibiotics susceptibility testing was done using Kirby-Bauer disc diffusion method. Phenotypic detection of ESBL in isolates was done by double disc synergy test (DDST). The ESBL genes were detected using conventional PCR method.
ResultsThe ESBL phenotypic positive isolates was (56.6 %). The most prevalent gene in the study was CTX-M gene. Antibiotic susceptibility of E. coli isolates to commonly used antibiotics was low. Isolates were most susceptible to quinolones (54.7 %) and fluoroquinolones (34.0 %). The ESBL producing isolates were more susceptible to quinolones but less susceptible to the third generation cephalosporins. There was significant association between gene expression by isolates and antibiotic resistance (P≤0.05). Isolates with the SHV and TEM genes showed 100 % resistance to some tested antibiotics. Isolates with CTX-M genes were also highly resistant.
ConclusionThe SHV, CTX-M and TEM genes were detected in Escherichia coli isolates in our locality. These may have resulted in the high resistance of isolates to commonly used antibiotics which may pose challenges to patient’s management.
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Investigating the effect of tobramycin dry powder inhaler on the eradication of Pseudomonas aeruginosa biofilms
More LessBiofilms are sessile communities of microorganisms embedded within a self-generated extracellular polymeric matrix. Such biofilms are found for instance in adults with cystic fibrosis, with pulmonary infections with the Gram-negative bacterium Pseudomonas aeruginosa being particularly common. This infection in CF patients is commonly managed with antibiotic dry powder inhalers, one of which is the aminoglycoside tobramycin. The activity of tobramycin has been well characterized in vitro, but current models that have been used are not very representative for lung infections, and better models would provide a significant advantage as these could be used, for instance, to improve the formulation of dry powder inhalers. For instance, one question that has not been addressed with current models is whether the size of drug particles emitted from a dry powder inhaler influences the efficacy of the anti-biofilm activity of the antibiotic. In this project, we utilized the Next Generation Impactor (NGI), which is a pharmaceutical instrument used to separate particles into size fractions. We used the NGI to separate tobramycin particles into different sizes and tested the influence of these particles on eradication of P. aeruginosa biofilms, which were grown using as colony biofilms that closely mimics conditions in the lung where biofilms are grown on a substrate-air interface. Preliminary evidence indicated smaller tobramycin particles are better in eradication of P. aeruginosa biofilms as compared to larger particles. Our results may represent a step towards improving the formulation of tobramycin dry powder inhalers to be effective in eradicating P. aeruginosa biofilms.
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Athlete’s foot: associated microbes and risk factors of infection transmission among football players
More LessBackgroundTinea pedis is one of the most common superficial skin infections and represents a major public health problem globally. It is common among athletes especially soccer players. This cross sectional prospective study was carried out to determine the degree of occurrence of tinea pedis and the associated risk factors among soccer players.
MethodsEighty subjects with visible lesion of tinea pedis were enrolled for the study after obtaining informed consent and Ethical clearance from the subjects and relevant authorities respectively. A structured questionnaire was administered to the subjects for data on risk factors for infection and demography. The AFSI was used to assess the lesions. Skin scrapings were obtained from lesions for analysis. Samples were subjected to microscopy, culture and physiologic testing.
ResultsA total of 52/80 (65.0 %) athlete’s foot infection rate was recorded in the study. Dermatophytes recovery rate was 29/52 (55.8 %) while yeasts and non-dermatophytes moulds’ recovery rate was 23/52 (44.2 %). Subjects with AFSI > 1 had (38.5%) infection rates but there was no significant association between AFSI and athletes’ foot (χ2=5.4; P≥0.05). Fungal and bacterial co-infection rate was 42.5 %. Trichophyton meantagrophyte 8 (15.5 %) was the most common dermatophyte while Aspergillus niger 6 (11.5 %) was the most common non-dermatophyte. The highest risk factor of infection transmission among subjects was the use of public gym 28 (35.0 %).
ConclusionDermatophytes and non-dermatophytes were associated with the athlete’s foot. The name tinea pedis should be reconsidered. The use of public sports facility may foster infection transmission.
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Construction and test of an efficient biophotonic imaging (BPI) reporter system to study pneumococcal biology in vitro and in vivo
More LessStreptococcus pneumoniae is a common nasopharyngeal resident in healthy persons, but remains a major cause of pneumonia, bacteremia and otitis media despite vaccines and effective antibiotics. There is an urgent need for novel therapeutic approaches, but such advances require a detailed knowledge of S. pneumoniae biology and its shift from commensal to pathogen. To better understand pneumococcal biology and infections, we need sensitive in vivo imaging technologies. To this end, bioluminescence imaging can be used, for example, to evaluate anti-infectives, intraspecies interaction and pneumococcal virulence non-invasively. A click beetle luciferase (CBR-luc) containing vector pPP3 under the control of putative highly expressed pneumococcal promoters was constructed. The CBRluc providing red-shifted light production was integrated into known sites in the S. pneumoniae genome. The constructs were compared to a lux-based exist system expressing bacterial luciferase using in vitro growth experiments. The results revealed that CBRluc tagged bacteria, PphrA::luc-wt, showed robust activity of bioluminescence in exponential phase that is maintained during stationary phase, whereas, lux-expressing pneumococci emitted a light signal with high background that peaked during exponential phase and was significantly reduced in intensity during stationary phase. Initial findings demonstrate that the CBRluc reporter system is more efficient than lux, providing a potential platform for utilization in understanding of the mechanisms of pneumococcal pathogenesis in vivo system.
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Antibiotic susceptibility pattern of Salmonella isolates from enteric fever suspected patients
More LessBackgroundEnteric fever is one of the most common diseases encountered worldwide and is endemic in Nepal. This study was conducted to access antibiotic susceptibility pattern of Salmonellaisolates from culture positive cases of enteric fever.
MethodsAltogether 505 blood samples were collected from patients clinically suspected of enteric fever attending HAMS Hospital. All blood samples were cultured by BACTEC method and sub cultured in blood agar and MacConkey agar plates. All isolates were identified by colony characteristics, biochemical tests and serotyping methods. Antibiotic susceptibility test was performed by modified Kirby Bauer disc diffusion method interpreted with CLSI guideline.
ResultIsolation rate of Salmonellaspecies was 3.6 %. Among 18 Salmonellaisolates, 10 were S. typhi, 8 were S. paratyphi A. The prevalence rate of infection was high among the age group 11–20 years (50 %) and among the male patients. However, there was no significant association of enteric fever with gender of patients (P=2.47). All 18 isolates were sensitive to Amoxycillin, Azithromycin, Ceftriaxone and Chloramphenicol, Ciprofloxacin and Ofloxacin. Majority of isolates were sensitive to Cefixime (94.4 %), Cotrimoxazole (94.4 %) and Cephotaxime (90 %). There were no any MDR isolates. Higher percentage of isolates was resistant to Nalidixic acid (87.5 %).
ConclusionThe decreased susceptibility to Fluroquinolones of S. typhi and S. Paratyphi A can be correlated with resistance to Nalidixic acid. Commonly used third generation Cephalosporins and rolled back first line drugs be the choice in case of NARS isolates.
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Antimicrobial activity of Zamzam water against Salmonella typhii in vitro
More LessObjectiveTo determine the antimicrobial effect of zam zam water against Salmonella typhi in vitro. The antimicrobial effect was measured from the minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of zamzam water against Salmonella typhi.
DesignThis experimental study used post-test only control group design with four time repetition. Step one was cultivating bacteria in liquid medium with various concentration of extract, that was 1 %, 1.25 %, 1.5 %, 1.75 %, 2 % with two control, extract control and bacterial control.
ResultsThe MIC (Minimal Inhibition Concentration) is 1.5 % concentration of extract. Step two was plating in NAP (Nutrient Agar Plate) medium. The MBC (Minimal Bactericidal Concentration) is 1.75 % concentration of zamzam water. The result of experiment was knew there are different average of Salmonella typhi colony from every group. The result experiment was analyzed by One Way Anova Test. The hypothesis test of MBC show significant differentiation, and then was continued with regression test. The conclusion of this study was zamzam water have antimicrobial activity against Salmonella typhii in vitro.
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Prevalence, susceptibility patterns and virulence factors of bacterial isolates from neonate-mother pair samples in Benin City, Nigeria
More LessNeonatal sepsis remains a major cause of morbidity and mortality in neonates. The prevalence of bacterial isolates in neonates admitted due to sepsis together with mothers and their susceptibility to routinely used antibacterial agents were investigated. Ethical Approval was obtained from the Hospital Management Board while informed consents were obtained from their parents. Forty-five (45) saliva samples from neonates (with signs and symptoms of sepsis admitted at the neonatal unit of Central Hospital, Benin City, Nigeria) were obtained. Vaginal swab samples were also obtained from their mothers to attain a total of ninety samples comprising forty-five saliva-vaginal swab sample pairs. Samples were immediately transported to the Laboratory and processed using standard microbiological protocols. All samples showed significant bacterial growth. At least one similar bacterial isolate were recovered from each neonate-mother pair. Bacteria isolated include Staphylococcus aureus, Klebsiella oxytoca, Klebsiella spp., Escherichia coli, Enterobacter spp., Proteus miriabilis, Acinetobacter sp., Proteus vulgaris, Pseudomonas aeruginosa, Streptococcus agalaticeae, Citrobacter sp. and Streptococcus pneumoniae. Both neonatal and maternal isolates were sensitive to unacin, azithromycin, cefotaxime and cefuroxime. Bacterial isolates also showed varying degrees of resistance to bactericidal action of normal serum. Isolates also produced haemolysin. This study gives important insight to the role of saliva in bacteriological analysis of sepsis and has implications for neonatal survival.
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Investigating the antimicrobial efficacy of MSCs as a potential novel therapy for Mycobacterium avium pulmonary infection
More LessNon-tuberculous mycobacterial pulmonary disease (NTM-PD) has rapidly increased in global prevalence over the last two decades. NTM-PD occurs mainly in patients with pre-existing structural lung disease and current treatment strategies are often ineffective and poorly tolerated. Outside of the cystic fibrosis population, the most common NTM isolates are from Mycobacterium avium complex (MAC). Mesenchymal stromal cells (MSCs) have potent antimicrobial and immunomodulatory properties including direct microbial killing and enhancement of phagocytic function. Their effect on MAC species is unknown. Human MSCs were infected with M. avium at a multiplicity of infection (MOI) of 2. Human monocyte-derived macrophages (MDMs) were also infected with a clinical isolate of M. avium at MOI of 2. After 4 h, MSCs were added at a ratio of 1 MSC:3 MDMs. After 24 and 74 h, colony counts were performed on supernatants and cell lysates. MSCs reduced total bacterial counts of M. avium by 24 % at 24 h (from 295×103/ml to 225×103/ml, P<0.05) and 40 % at 72 h (from 403×103/ml to 243×103/ml, P<0.05). MSCs reduced total bacterial counts of M. avium in infected MDMs by >40 % (from 381×103/ml to 209×103/ml, P<0.05) after 24 h and >70 % after 72 h (from 1050×103/ml to 314×103/ml, P<0.05). MSCs have modest direct antimicrobial effect against MAC, but potently enhance their killing by macrophages. Mechanistic studies are required to understand the mechanisms of the antimicrobial effect, with the aim of exploiting these therapeutically in pulmonary MAC disease.
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Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens identifies isogenic strains in multiple outbreaks and novel virulence-related features
BackgroundClostridium perfringensis a major enteric pathogen known to cause gastroenteritis in humans. Although major outbreak cases are frequently reported, to date no Whole Genome Sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of C. perfringens-associated outbreak strains.
MethodsWe have applied both genomic and phylogenetic analyses on a sub-set of 109 newly-sequenced C. perfringens strains isolated from gastroenteritis-associated disease cases (including food-poisoning and care-home diarrhoea) from England and Wales between 2011 – 2017 to probe virulence profiles including toxin and AMR genes, plasmid features and genomic epidemiology of these case isolates.
ResultsOur data identified that highly-similar C. perfringens strains were associated with 9 care home-associated individual outbreaks over a 5 year interval, indicating potential common sources linked to these distinct outbreaks. Enterotoxin gene cpe was encoded in all but 4 isolates (96.4 % type-F strains), and it was further determined that virulence plasmids encoding cpe were extensively distributed in the isolates (97 % care-home isolates carry pCPF5603 plasmid; 60 % food-poisoning isolates carry pCPF4969 plasmid). Further virulence factors, such as β2-toxin, were enriched in these isolates (46.7 %). Phage proteins were also commonly identified, with additional analysis indicating phages may contribute to spread of virulence determinants.
ConclusionThis study highlights the genotypic and epidemiological relatedness of a large collection of C. perfringens strains isolated from gastroenteritis-associated cases from across the UK and Wales. Key points revealed include the potential circulation of disease-associated strains, and impact of cpe-encoding-plasmid disseminations, linked to outbreak cases.
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Unraveling the role of C. difficile S-layer in infection and disease
More LessClostridium difficile is a Gram-positive spore-forming bacterium. Gut colonization is associated with a wide spectrum of gastrointestinal diseases, ranging from mild to severe, acute to persistent. Bacterial survival and multiplication are essential processes dependent in the maintenance of homeostasis, which is, in turn, inherently dependent on the integrity of the cell wall. Bacteria present different mechanisms to maintain cell wall integrity, particularly, to protect it from direct contact with the external environment, such as phosphate polymers, capsule, outer membrane and S-layer. The S-layer is an evolutionary conserved macromolecule present in almost all bacterial types, however its role(s) remain(s) to be clarified. The S-layer is ubiquitous in C. difficile strains and is comprised of a conserved and a variable region that confers strain specificity and faces the external environment. In C. difficile FM2.5 strain, the absence of S-layer resulted in impaired pathogenicity. Gut colonization of FM2.5 infected mice was significantly lower than mice infected with the wild-type strain R20291. In addition, FM2.5 showed compromised toxin activity and in vitromotility. Accordingly, FM2.5 failed to cause disease in mice and trigger a strong inflammatory response, contrary to mice infected with R20291. Reversion of the S-layer gene restored motility, toxin activity and the abilities to colonize and cause disease. It appears that the S-layer plays a crucial role in various bacterial processes, hence FM2.5 loss of virulence may be due both to the absence of S-layer at the cell surface and its role in other processes that aid to bacterial virulence.
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Intracellular survival of Enterobacter cloacae complex in human macrophages
More LessThe Enterobacter cloacae complex (Ecc) is a group of enteric Gram-negative bacteria that also exist as commensals in nature. Ecc bacteria are also responsible for nosocomial outbreaks, targeting primarily immunocompromised patients and causing a wide range of systemic infections. Infection is often fatal as management is complicated by the high-level multidrug resistance expressed by Ecc isolates. Indeed, the Enterobacter species represent the last ‘E’ of the ESKAPE pathogens, which are the global leading causes of nosocomial infections. Despite the clinical relevance of Ecc, little is known about their virulence-associated properties and pathogenicity. Bridging this gap in knowledge is fundamental to provide a blueprint to better tackle Ecc infections. The aim of this study is to explore how E. cloacae interact with human macrophages and identify the bacterial and host cell factors involved in this process. Differentiated, human THP-1 monocytic macrophages were infected with fluorescent E. cloacae for various lengths of time. Macrophage infection was analysed by confocal microscopy and images processed on ImageJ and LAS X. Data suggests that approximately 90 % of intracellular E. cloacae are killed by 5 h post-infection, but the remaining 10 % of the bacterial population survives and persists up to 48 h post-infection. We have also observed E. cloacae colocalising with early and late endosomes as well as lysosomes. Our current results suggest that E. cloacae can subvert normal phagocytic trafficking and possibly adapt to survive inside the acidic lysosomal environment.
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Bovine tuberculosis in Eastern Ethiopia: prevalence, risk factors and its public health importance
Bovine tuberculosis is among the primary zoonotic disease caused by Mycobacterium bovis. A cross-sectional study was conductedon 315 cattle in selected areas of eastern Ethiopia, aiming to estimate the occurrence of bovine tuberculosis using comparative intradermal tuberculin skin test and assess cattle owners’ awareness on its public health implication. Random sampling method was applied in order to select animals from farm/household. Forty three farm/household owners of tuberculin tested animals were interviewed using pre-tested structured questionnaires. The overall prevalence of bovine tuberculosis was 20.3 % (n=64) in dairy cattle at recommended cut off >4 mm. From a total of 43 farms/households tested, 22 were positive; each farm exhibited at least one tuberculin positive reactor animal with a total herd level prevalence of 51.2 %. The prevalence of bovine tuberculosis in individual animal level was significantly different (χ2=45.2; P-value <0.001) in different sites. Farming system, herd size and other risk factors were significantly (P<0.05) associated with bovine tuberculosis occurrence. Of the total interviewed farm owners, only 33 % had the knowledge of or had heard about bovine tuberculosis and 23 % respondents were aware of the zoonotic importance of the disease. More than 50 % of the interviewees had shown their preference of raw milk consumption. The study showed bovine tuberculosis is highly prevalent. The majority of cattle owners lack awareness about the disease and its public health significance. Awareness rising about the disease, its transmission and zoonotic implication is of great importance for reduction and control measures.
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Occurrence and evaluation of antimicrobial susceptibility of Staphylococcus aureus isolated from chicken eggs, Eastern Ethiopia
More LessStaphylococcus aureus is responsible for a variety of infections in humans and animals that particularly causes staphylococcal food poisoning when it present in foods. This study was aimed to isolate Staphylococcus aureus present on the shell surfaces and in the contents of chicken eggs, and determine antimicrobial susceptibility patterns. A total of 335 egg samples were obtained from open market (n=174) and poultry farm (n=161). A sterile cotton swab was used to sample the surface of eggs. After sterilizing the shells, the egg contents were sampled. Isolation of Staphylococcus aureus was done based on ISO. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of the total 335 eggs sample examined, 93 (27.8 %) samples yielded S. aureus. Out of these, 28 (17.4 %) were from poultry farm while 65 (37.4 %) were obtained from open market. Similarly, 63 (18.8 %) were from the shell while 30 (8.9 %) were from the content. The level of S. aureus in egg contents was significantly higher in the open market (CI=0.0962–0.6085; P=0.003). All 76 S. aureus isolates were resistant to at least one of the antimicrobials tested with overall 3.9–92.0 % level of resistance pattern showing higher resistant to penicillin (92 %), and ampicillin (89.5 %). Multiple drug resistance was detected in 86.8 % of the total S. aureus isolates. The study showed high level of S. aureus with considerable antimicrobial resistant pattern. Further study is needed to better define bacterial resistance to antimicrobial agents with emphasis of multiple drug resistant.
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Selection of specific peptides recognised by polyclonal antibody from Salmonella enteritidis infected chicken using next generation phage display technology
More LessSalmonella Enteritidis is an important cause of human salmonellosis and food-poisoning associated with consumption of contaminated chicken eggs and poultry products. Objective of the study was to develop a serological diagnostic testfor the recognition of different Salmonella serovars in chickens. Here, phage peptide libraries were screened against 9 IgY samples from chickens infected with S. Enteritidis and 9 IgY samples with S. Hadar and the individual peptide binders were then identified using NGS. Twenty-nine peptides were identifiedin silico assessmentas being enriched specifically against IgY from multiple chickens infected with S. Enteritidis compared to those infected with S. Hadar. Twenty Nine peptides identifiedin silico assessmentwere thentested by both training and test cohorts of chicken IgY samples in ELISAs. The training set of samples was made upof IgY from 9 chickens infected with S. Enteritidis and 9 infected with S. Hadar. Seventeen peptides were selected as the most recognized specific peptides against S. Enteritidis infection and were then used against IgY samples from10 birds infected with S. Enteritids and20 birds with S. Typhimurium as a test cohort. Overall, for both training and test cohorts the peptide ELISA assay sensitivity and specificity were 90 % for detecting infections. The most discriminatory peptides by ELISA test were AEGEFEPQSARPS and AEGEFFVNRALINQ. The data demonstrated that the NGPD method could identify peptides that represented serovar-specific epitopes/mimotopes, these peptide have potentially important applications for the development of peptide based immuno-diagnostic assays for the recognition of Salmonella Enteritidis in chickens.
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Molecular epidemiology and antimicrobial resistance of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in the UK between 2004-2017
The genus Corynebacterium includes three potentially toxigenic species, C. diphtheriae, C. ulcerans and C. pseudotuberculosis, capable of causing diphtheria, a severe disease in humans. The aims of this study were to undertake Multi-Locus Sequence Typing (MLST) on a panel of both toxigenic and non-toxigenic C. diphtheriae and C. ulcerans strains (20 toxigenic and29 non-toxigenic C. diphtheriae, 17 toxigenic and 14 non-toxigenic C. ulcerans) isolated in the UK between 2004 and 2017, and use these results to determine the molecular epidemiology within the UK. Antibiotic sensitivity testing was also undertaken (20 toxigenic and 88 non-toxigenic C. diphtheriae, 17 toxigenic and 14 non-toxigenic C. ulcerans) and their profiles compared. The MLST results showed that C. diphtheriae and C. ulcerans isolates formed two distinct genetic populations and that C. diphtheriae isolates with intermediate penicillin resistance demonstrated sequence types which were genetically related. The results also showed that ST32 was most prevalent (31%, 9/29 isolates) amongst non-toxigenic C. diphtheriae. Non-toxigenic C. ulcerans isolates demonstrating intermediate penicillin resistance formed distinct genetic populations and appeared distantly related or unrelated. There were 75 % (15 isolates) of toxigenic C. diphtheriae isolates, 35 % (6 isolates) of toxigenic C. ulcerans isolates, 30 % (26 isolates) non-toxigenic C. diphtheriae and 43 % (6 isolates) of non-toxigenic C. ulcerans which demonstrated intermediate penicillin resistance. Linezolid and vancomycin were the only antibiotics which demonstrated 100 % sensitive profiles for all isolate groups. These data will help inform public health guidance and management of corynebacteria infections caused by these species.
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Antimicrobial resistance in non-O157 Shiga-toxin producing Escherichia coli
More LessObjectivesShiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens that cause severe gastrointestinal disease in humans. Monitoring antimicrobial resistance (AMR) in STEC from symptomatic human cases may provide evidence for the extent of transmission of resistant strains and resistance genes from ruminants to humans. The aim of this study was to assess AMR in non-O157 STEC in England and Wales between 2014 and 2016, and to compare phenotypic and Whole Genome Sequencing (WGS) derived AMR profiles.
MethodsSix hundred and fifty-three non-O157 STEC isolates were analysed. WGS and bioinformatic analysis were performed on 457 isolates in the top 10 Clonal Complexes (CC) (193 were excluded on the basis of CC) and phenotypic susceptibility typing via breakpoint and minimum inhibitory concentration testing was undertaken on 100 isolates exhibiting resistance to at least one antimicrobial.
ResultsOf 457 isolates, 332 lacked identifiable resistance genes and were predicted to be fully susceptible to 11 diverse classes of antimicrobials, 125 were found to carry one or more resistance genes and 83 were multi-drug resistant. Four isolates were identified as extended-spectrum b-lactamase-producers. In total, 46 different genes were detected – which conferred resistance to 8 different antibiotic classes. An overall concordance of 97.5 % was demonstrated between the two methods.
ConclusionsPhenotypic and genome-derived AMR comparisons showed good correlation for non-O157 STEC. This has added to the evidence base to support the use of genotypic approaches for antimicrobial susceptibility typing, to replace phenotypic typing for surveillance purposes, and guide clinical decision making in the more distant future.
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Augmenting Staphylococcal infection: the importance of timing
More LessStaphylococcus aureus (S. aureus) acts as a commensal in the microbiome of the skin and nasopharynx. However, on gaining access to the bloodstream it can cause an array of pathogenic outcomes. S. aureus can crowdsource the microflora to assist in becoming an opportunistic pathogen as our lab has recently published findings that co-inoculation of S. aureus with commensals, acting as pro-infectious agents, leads to a much more robust, virulent infection. This benefits S. aureus at doses where it would otherwise be cleared by the immune system. Pro-infectious agents do not need to be live commensals as isolated cell wall peptidoglycan also augments infection. This work aimed to assess the effects of inoculation with pro-infectious agents before and after infection with S. aureus. It was found that pro-infectious agents needed to be co-administered in order to fully augment infection. This gives mechanistic insight where S. aureus and the pro-infectious agents need to be in the same local environment or phagocyte to augment infection.
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Clinical evaluation of the novel molecular bacterial load assay for real-time monitoring of tuberculosis treatment response
More LessBackgroundMonitoring the treatment of tuberculosis (TB) relies on less sensitive smear microscopy (SM) and culture methods which are very slow. We evaluated the novel Molecular bacterial load assay (MBLA) for implimentability and real-time monitoring of TB treatment in a clinical setting.
MethodsTherapy naive (Xpert MTB/RIF confirmed) TB positive patients were enrolled in Mbeya, Tanzania. Sputum samples were collected at baseline and thereafter at week 2, month 2, 5 and 6 of treatment. Samples were analysed for M. tuberculosis (M.tb) by MBLA and compared to SM, culture and clinical monitoring.
Results59 TB patients were enrolled for the study. Median age, 37 (18-65) years, 62.7 % (37/59) male, 45.6 % (27/59) HIV positive and 8.47 % (5/59) were re-treatment. Mean BL (± SD) at baseline was 5.48 ± 1.3 declining to 3.42 ± 0.7 at month 2 and 3.51 ± 0.62 log10CFU/ml at month 6 of treatment. This corresponds MBLA positivity of 92.98, 65.5 and 7.84 % at baseline, month 2 and 6 respectively. In contrast, positivity of SM and culture were 78.95, 9.62 and 0 %, and 85.96, 25 and 3.39 % at baseline, month 2 and 6 respectively. Decline in test positivity reflected resolution of clinical signs. While night sweat, and chest pain resolved earlier on in treatment, resolution of cough was slow and consistent with MBLA. Furthermore, the turn-around-time for MBLA results was 24 h compared to median (range) of 14.83 (4.33–42) days for liquid culture.
ConclusionMBLA exhibited higher sensitivity and shorter turn-around-time than standard tests and clinical signs. This demonstrates the potential of MBLA to offer real-time results for clinical decision making.
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Mapping 4D pH evolution in Streptococcus mutans biofilms using fluorescent ratiometric pH-sensitive nanosensors
More LessStreptococcus mutans is a pre-dominant bacterial species found in oral biofilms and participates in the production of dental caries via the generation of organic acids. The production of these acids results from the fermentation of carbohydrates present in a sugar-laden diet. As the acidity of an oral biofilm decreases, the demineralisation of the enamel of a tooth increases; leading to the formation of dental caries. To detect and measure the pH change occurring following a sugar challenge, ratiometric, fluorescent, pH-sensitive nanosensors were incorporated into oral biofilms. Confocal laser scanning microscopy revealed that the addition of glucose (1 % w/v) to an S. mutans biofilm resulted in a gradual reduction in the fluorescence intensity ratio during a 30 min period. This reduction in the fluorescence intensity ratio indicated a reduction in pH of the biofilm over time as the glucose was being fermented, resulting in the production and secretion of acids into the extracellular matrix of the biofilm. Additionally, a reduction in pH was detected – using widefield microscopy – in starved, planktonic S. mutans when treated with glucose. Over the course of 30 min, the pH of the medium was reduced from pH 5.3 to pH 3.3 as the glucose was fermented by the bacteria. These findings will help us map pH changes in oral biofilms as we examine potential methods of preventing the acidification of oral biofilms and the eventual demineralisation of the enamel; leading to the reduction in dental caries and an improvement in the standard of living of those effected.
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The O6 antigen of Escherichia coli strain CFT073 is a target for Myoviridae
More LessExtraintestinal pathogenic Escherichia coli strain CFT073 is a prototypic urosepsis isolate. CFT073 is of serotype O6:K2:H1 and thus has an O-antigen. The wzy gene encodes the O-antigen polymerase Wzy, which catalyses the polymerisation of O subunits into a long chain polysaccharide. A CFT073 Δwzy mutant was constructed using the λ Red recombination system. The lack of O-antigen was confirmed by LPS purification and staining with Pro-Q Emerald 300. CFT073 Δwzy and wild-type CFT073 were tested for their bacteriophage sensitivity. ΦEB49, a member of the Myoviridae, can mediate generalised transduction in strain CFT073. CFT073 Δwzy and wild-type CFT073 mixed with the same concentrations of ΦEB49 phage were compared. Confluent growth was evident on all CFT073 Δwzy plates, whilst lysis was apparent on all CFT073 plates. The inability of ΦEB49 to lyse CFT073 Δwzy suggested that this phage binds to O antigen, hence the decreased O antigen of CFT073 Δwzy, compared to wild-type CFT073, prevented phage lysis from occurring. These data indicate that CFT073 Δwzy is not a susceptible host for ΦEB49.
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A micro luminescence-based assay to measure serum susceptibility in Escherichia coli
More LessNeonatal meningitis Escherichia coli (NMEC) is the leading cause of gram-negative meningitis in neonates, and it contributes to neonatal morbidity and mortality globally. The prototypic strain of NMEC, E. coli RS218 possesses the K1 capsule and has been widely employed in the study of NMEC pathogenesis. Previously, our laboratory has utilised relatively large volumes of culture to assay serum bactericidal activities, to garner valuable insights into bacterial immune evasion strategies. However, these methods can be labour intensive, time consuming and are not easily adaptable for high throughput applications. To overcome these limitations, a smaller volume real-time assay was developed. Bacteria were cultured in 100 µl volumes and were sub-inoculated for logarithmic growth. A slow kinetic absorbance assay was established on the Optima Fluorostar microplate reader, which enabled the accurate and reliable measurement of bacterial growth. Subsequently, bioluminescence was incorporated into the assay to facilitate the measurement of bacterial viability in real-time. Bacteria were rendered bioluminescent via electroporation of the pIlux plasmid or alternatively by the addition of exogenous beetle luciferin and recombinant firefly luciferase. The utility of these approaches in the determination of bacterial complement evasion is reported below.
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Intracellular replication of pneumococcus in ex vivo-perfused human spleens
Recently, we have demonstrated that the pathogenesis of Streptococcus pneumoniae bacteraemia contains a concomitant phase of intracellular replication within splenic macrophages in mice (Ercoli et al. Nat Micro. 2018). In the present study, we aimed to determine if intracellular replication of pneumococci may play a role in the pathogenesis of sepsis in humans, using two innovative approaches. We used a model (Chung et al. ALTREX.2018) involving ex vivo perfusion of human spleens from elective splenectomy patients (REC reference: 18/EM/0057). Organs were infected with 6.5×107 c.f.u. of pneumococci, and serial biopsies and ‘blood’ samples were taken at predetermined times. Samples were analysed by colony counts, confocal microscopy and flow cytometry. Additionally, infected tissue samples were taken for preparation of organotypic slice culture time courses. Bacteria injected into the perfusion circuit were rapidly cleared at early time points post-infection, recapitulating what is observed in experimental murine sepsis. Bacterial counts in the spleen increased, providing initial evidence of intracellular bacterial persistence. Microscopy analysis indicated that bacteria could be localised to splenic macrophages, with the size of infectious foci increased over time. Z-stack microscopy localised bacteria within cell membranes, indicating the infection was predominantly intracellular. In ex vivo slice cultures increasingly large numbers of pneumococci were cultured over time, further indicating intracellular replication. In conclusion, we provide evidence for a role of intracellular replication of pneumococci in human splenic macrophages in the pathogenesis of sepsis.
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Characterization of a bacteriophage from avian Staphylococcus aureus associated with innate immune evasion
More LessStaphylococcus aureus is an important human and livestock pathogen. An S. aureus phage (φAvβ) inserted into the chromosome at the beta toxin gene (beta-converting phage) is present in approx. 90 % of human strains and contributes to human-specific innate immune evasion. Comparative genomic analysis of S. aureus isolates from infected poultry has revealed an avian-specific subfamily of beta-converting phages represented by multiple variants with distinct integrase gene alleles. To investigate the role of the avian beta-converting phages in host-pathogen interactions, a φAvβ-deficient strain with non-functional beta-toxinand a φAvβ-deficient strain with restored beta-toxin were constructed by allele replacement in an avian pathogenic S. aureus strain. Compared to the wild type, both φAvβ-deficient strains have reduced net extracellular growth in vitro in chicken bone-marrow derived macrophages. Further investigation using GFP-tagged bacteria has revealed that both φAvβ-deficient strains show reduced initial phagocytosis and intracellular survival compared to the wild type. Absence of φAvβ is also associated with decreased killing of the chicken bone-marrow derived macrophages. We are currently investigating the mechanism underlying this phenotype using deletion mutants of the candidate phage effector genes. Ongoing work also involves using RNAseq to investigate differential host transcriptional response of the macrophages to S. aureus in presence/absence ofφAvβ. Overall, these data will contribute new information relating to the evolution of avian S. aureus and mechanisms of bacterial host-adaptation.
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Improved molecular typing of toxigenic Clostridium difficile strains affecting animal and human health
More LessClostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.
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Detection and quantification of Staphylococcus aureus heterogeneity to identify antibiotic-induced persistence
More LessPersister cells are characterised as being viable but non-culturable, a state that preserves their metabolic energy to survive the environmental stress, which allows for recurrent infections. Detection of persisters is, therefore, not possible with standard culture-dependent methods. Furthermore, the effect of antibiotics on the induction of persisters has not been assessed. This study aimed to identify antibiotic-induced persistence and determine the percentage of heterogeneity. Vancomycin, daptomycin and dalbavancin were assessed by standard MIC methods against selected Staphylococcus aureus strains. Replicates of MIC assays were stained with propidium iodide to quantify live/dead and a reactive oxygen species (ROS) dye to detect and quantify persisters using culture-independent single-cell sorting, independently. A comparative analysis was then performed. Dalbavancin showed the lowest MIC values against tested S. aureus strains followed by daptomycin and vancomycin. Cell sorting of vancomycin-, daptomycin- and dalbavancin-treated S. aureus strains showed a range of 1.9–10.2 %, 17.7–62.9 % and 7.5–77.6 % live cells based on the strain, respectively, in which daptomycin, in particular, was a strong inducer of a persister population. Persisters represented 3.7–16 % of the bacterial population. The culture-independent identification of antibiotic-induced persistence through studying at the single-cell level showed different efficacy of antibiotics than standard MIC. Vancomycin was the most effective antibiotic against tested strains followed by dalbavancin then daptomycin as assessed by cell sorting. Therefore, re-evaluation of standard MIC methods may be required to assess the efficacy of antibiotics. Additionally, the detection of daptomycin-associated persisters may provide an elucidation to the reported rapid resistance development in vivo.
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In vitro antimicrobial efficacy of Cnidoscolus aconitifolius leaf extract and honey on Staphylococcus epidermidis clinical isolate
More LessThe search for alternative, potent, cost-effective treatment of ailments caused by resistant microorganisms and the role of plants and their products as essential sources of medicinal agents is receiving increasing attention. Several of these natural products are reported to have capacity to produce natural compounds of high structural diversity that serve as defense agents against invading microorganisms. Cnidoscolus aconitifolius also known as tree spinach is an indigenous tropical tree that has gained lot of importance in its nutritive value and traditional use. This study evaluates the possible effect of Cnidoscolus aconitifolius leaf extract alone, extract/honey combination on Staphylococcus epidermidis clinical isolate. Phytochemical analysis and antimicrobial efficacy of the extract were performed using standard methods. The results of phytochemical analysis reveal the presence of carbohydrate, tannins, alkaloids, steroids, flavonoids, anthraquinone, saponins and carotenoids. Antimicrobial activities showed inhibition zone values of 8.0±0.1 mm for aqueous extract alone, 9.0±0.1 mm for aqueous extract/honey combination. The finding suggests that C. aconitifolius might be a good source of compounds that can be used to inhibit the growth of Staphylococcus epidermidis pathogen and further supports its popular and wide traditional applications in the treatment of various illnesses. Hence the need for further research to exploit the full potential of C. aconitifolius tree in order to influence their extensive consumption, storage, improvement and production.
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Novel antimicrobial agents for clinical applications
More LessSince their discovery, antimicrobial compounds have been vital for the treatment and prevention of disease; making many previously fatal diseases treatable or at worst, manageable conditions. The inappropriate use of these compounds has led to the rapid development of resistance mechanisms within bacteria to the majority of compounds currently marketed. A recent UK governmental review predicted that by 2050 global deaths caused by antimicrobial resistant bacteria will outnumber those attributed to cancer [1]. As new resistance mechanisms emerge and resistance within microbial populations increases, so does the need to further understand the molecular basis of resistance, develop new antimicrobial molecules and use better strategies to manage their use [2]. In response to this, we discovered a novel class of antimicrobials and have created 50 structurally related members of this class [3-6]. We sought to understand the structure-activity relationships which will result in the determination of the mode of action of these molecules. Consequently, each variant was screened against Staphylococcus aureus and Escherichia coli and the minimum inhibitory concentration was calculated for effective compounds. This will enable us to identify predictive tools that will aid the synthesis of the next generation of these novel therapeutic molecules. We will present our latest findings in the ongoing analysis of the antimicrobial activity for each variant of this new class of antimicrobial compound. In addition, we will discuss the insights provided by the detailed structure-function analysis. This project is in collaboration with Public Health England and NHS East Kent Trust.
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Prevalence of extended spectrum beta-lactamase producing Enterobacteriaceae in urine samples from Thumbay hospitals, U.A.E
Beta Lactamases is proven to be one of the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria. Many up to date researches have shown increase in the incidence and prevalence of ESBL worldwide. This study aimed to determine the prevalence of ESBL strains of Klebsiella spp. and Escherichia coli species in urinary isolates from the patients admitted in Thumbay hospitals around United Arab Emirates. Furthermore, drug resistant genes (SHV and CTX-M) in the ESBL positive samples were detected. 237 urine samples were collected from November 2017 to January 2018. Based on the lactose utilization, colony morphology, and biochemical utilization of the gram negative bacilli were identified as E. coli (53), Klebsiella pneumoniae (10) and Citrobacter species (2). Antibiotic sensitivity test, double disc diffusion test and combination disc tests all confirmed that the 65 (27.4 %) out of 237 isolates were ESBL producing bacteria. There was high prevalence of bacteria in females than male and the number of E. coli strains is higher than Klebsiella spp. DNA isolation was performed on the 65 samples, out of which 50 samples were selected for PCR based on their concentration. The selected DNA samples were used to detect the presence of bla CTX-M and bla SHV genes. Only 24 DNA samples (48 %) contains blaCTX-M genes, bla SHV or both the genes. 14 samples had bla CTX-M gene, 2 bla SHV genes, and 8 with both bla SHV and bla CTX-M. At the rate at which ESBL is spreading, further research, close observation and cautious use of antibiotics is important.
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Harnessing novel bacterial peptides for antimicrobial activity in the gut microbiome
More LessThe Enterococci are a resilient collection of species, found in the human intestine, river sediment and even certain cheeses. Human infection by this genus is dominated by E. faecalis and E. faecium. Vancomycin resistant enterococci (VRE) are associated with higher mortality rates over non-VRE strains. Enterococci can utilise the highly efficient pheromone responsive plasmid (PRP) system to transfer plasmid DNA between cells. Plasmid containing donor cells respond to small peptide pheromones (7–8 amino acids) and transfer plasmid DNA to pheromone-producing plasmid-free recipient cells. PRP can encode antibiotic resistance (including vancomycin) and virulence enhancing factors. Investigation into the PRP system between donor and recipient E. faecalis environmental isolates has indicated a 40 % decrease in PRP transfer in colder environments. Additionally, PRP efficiencies under other conditions, including in presence of synthetic pheromone peptides, have been calculated. Future assays will utilise pheromone imitative fluorescently labelled synthetic peptides to visualise the pheromone binding receptor (PrgZ) on the E. faecalis donor cell membrane. Later experiments will focus on varying the synthetic pheromone amino acid composition so to interfere with the PRP system machinery, with the aim of reducing PRP transfer efficiency or preventing PRP transfer completely.
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Application of furanone compounds for the modulation of biofilm formation in common wound pathogens
More LessChronic wounds are a significant issue in healthcare, presenting a considerable economic burden to the NHS and a serious health risk to patients. The majority of non-healing wounds have been shown to contain a biofilm which prolongs the inflammatory stage of wound healing and significantly delays wound healing. This often causes a normal wound to progress and become chronic, presenting further problems for patients including increased risk of secondary infection, further deterioration of the wound and an increase in treatment intensity. This project aims to assess the efficacy of several compounds in modulating the formation of biofilms in a number of clinically relevant pathogens when used at sub-inhibitory concentrations. The organisms used in this project include Pseudomonas aeruginosa and Staphylococcus aureus. We aim to test the efficacy of three plant derived compounds including 4-hydroxy-2,5-dimethyl-3(2 h) furanone (HDMF), 2-methyltetrahydrofuran-3-one (MTHF) and l-ascorbic acid. Future work will characterise the efficacy of these compounds when delivered to a biofilm from a hydrogel based delivery system. At sub-inhibitory concentrations in pure solution, candidate molecules tested to date showed no ability to reduce biofilm formation. Indeed, treatment with HDMF resulted in greater production of biofilm in P. aeruginosa and treatment with all compounds showed no difference in biofilm formation by S. aureus. To characterise the impact of hydrogel based delivery on compound efficacy all candidate molecules were loaded into a hydrogel and shown to be effectively released from it. Experiments to characterise the modulatory potential of these compounds when released from a hydrogel are currently underway.
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The effect of local release antibiotic beads on in-vitro bacterial growth from tissue taken from infected diabetic foot ulcers
More LessDiabetic foot infection is the main reason for diabetes-related hospitalisation and is a major cause of diabetes-related amputation. Recent figures published by Public Health England show that there are more than 163 diabetes related amputations in England every week. This study investigates the effect of antibiotic loaded calcium sulfate (Stimulan® Rapid Cure) beads on in-vitro bacterial growth from tissue taken from diabetic foot infections. Patients were recruited from the Macleod Diabetes and Endocrine Centre at the Royal Devon and Exeter Hospital. Inclusion in the study was based on clinical recognition of an infected foot ulcer requiring wound debridement. Debrided tissue was homogenised and 50 µl spread over the surface of Columbia blood agar and fastidious anaerobe agar. Three replicate calcium sulfate beads containing a combination of vancomycin and gentamicin were then placed on the surface of the agar. Each bead contained approximately 3.4 mg and 1.6 mg of vancomycin and gentamicin respectively. Plates were incubated aerobically or anaerobically as appropriate. Zones of inhibition were recorded at 1 and 4 days. Calcium sulfate beads containing vancomycin and gentamicin were able to inhibit bacterial growth in all tissue homogenates tested with zone diameters ranging from 16 to 40 mm. Local release of antibiotics could have the benefit of achieving high local concentrations within poorly vascularised tissue which may inhibit bacterial growth at the wound site. By improving treatment of diabetic foot infections, it may be possible to prevent amputation, maintain mobility and conserve quality of life.
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Epidemiological characteristics of the Manchurian plague pandemic of 1910-1911
More LessObjectiveTo investigate the epidemiological characteristics of the Manchurian plague pandemic of 1910–1911 with descriptive epidemiological methods.
MethodsThe epidemiological data were distracted from Report of Manchurian Plague, which is a summary of local official reports about plague in 1910–1911. The time distribution by day, and the space distribution by country was recorded, and also the source of infection.
ResultsThe pandemic of plague continued from October 25th, 1910 to April 18th, 1911. There were 46 747 dead cases on record in the three provinces of Manchuria. There were 24 867 dead cases and 7068 dead cases were reported, and the average mortality rates were 41.21 and 10.25 per ten thousands in Jilin and Liaoning province, respectively. In Heilongjiang province, 14 812 dead cases of plague were reported. The huge difference was found in different epidemic regions, the highest mortality rate was 4121 per ten thousands in Binjiang country of Jilin province. Patient zero of pneumonic plague had been infected in Russia and got sick and died in Manzhouli, a northern country in Heilongjiang province. Then the pneumonic plague was mainly spread through railway to other cities.
ConclusionThe epidemiological characteristics of the Manchurian plague pandemic of 1910–1911 were first described with modern epidemiological methods.
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Detection and characterisation of bacteria causing lung infection in people with Cystic Fibrosis (CF) by surface-enhanced Raman spectroscopy (SERS)
More LessRapid and accurate identification of pathogens in CF could ensure prompt treatment with the most appropriate antibiotic; potentially improving outcomes and shortening hospital stays. As traditional culture methods for detecting bacteria are time-consuming there is a growing interest in SERS as a novel culture-free technique that produces a whole-organism spectroscopic fingerprint at high speed. Bacterial isolates including Pseudomonas aeruginosa (n=32), Staphylococcus aureus (n=5), Streptococcus pneumoniae (n=5) were incubated for 6 h at 37 °C/180 r.p.m., with a starting optical density (OD) of 0.15. After adjustment of the OD to 0.3, bacterial cells were harvested by centrifugation at 9000 rcf for 3 min and washed three times with dH2O. Bacterial pellets were mixed with citrate reduced silver colloid (CRSC) and dried. Spectra were recorded (4×10 s at 785 nm) and analysed within GRAMS/Al using Principal Component Analysis (PCA). Spectra of P. aeruginosa isolates (n=32) were separated into two distinct groups; the spectra of one group (n=12) was dominated by the pigment pyocyanin with vibrational brands present at 1350, 1492, 1598 and 1615 cm-1. The other group (n=20) had characteristic vibrational bands at 661, 735 and 800 cm-1 which correspond to guanine, adenine and uracil, respectively. S. aureus has a main characteristic band at 735 cm-1 and S. pneumoniae has a characteristic band present at 480 cm-1. Bacterial species clustered separately when analysed by PCA. Reproducible and distinguishable SERS spectra of bacterial isolates were obtained, and it was possible to differentiate between different bacterial species using PCA. These results suggest SERS has the potential to rapidly detect bacteria.
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Difficulties in diagnosis and treatment of urinary tract infections in an elderly population
More LessBackgroundUrinary tract infection (UTI) contributes significantly to healthcare burden, accounting for 23 % of hospital acquired infections and 2–3 % of general practice consultations. Unfortunately, difficulties exist in obtaining an accurate diagnosis, with studies showing misdiagnosis rates above 40 % in elderly populations. Furthermore, numerous hospitals across the UK still advocate the use of Trimethoprim for UTI, despite high rates of resistance. These factors combined leads to a sub-optimal experience for patients.
AimWe aimed to identify the practices surrounding the diagnosis and treatment of UTIs in elderly patients within Royal Bolton Hospital, a large district general hospital in the North-West of England. We also aimed to identify unique patterns of presentation of UTIs in elderly patients which could lead to diagnostic difficulty. Finally, we assessed local antibiotic resistance rates.
MethodsA retrospective case-note analysis of 100 patients, over the age of 65 years, diagnosed with UTI was carried out in 3 cycles between 2016–2018. The final cycle was conducted following removal of Trimethoprim from antibiotic guidance.
ResultsOf patients diagnosed with UTI and had MSU (mid-stream urine) sample analysed, only 28.8 % displayed microbial growth. 39.1 % of patients with confirmed UTI displayed neither signs nor symptoms of UTI. 20 % diagnosed with UTI did not have a MSU sample requested. Resistance rates of 39.1 % were reported to Trimethoprim, with E. coli accounting for 56.5 % of all UTIs.
ConclusionsDiverse presentation and incomplete diagnostics contributes to misdiagnosis of UTI. Trimethoprim is not an effective treatment option and guidelines should reflect this.
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Shining a light on antibiotic selection: optimised live/dead fluorescence spectrometry for rapid antimicrobial susceptibility testing
More LessAntibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We propose to use live/dead spectrometry as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We have developed a spectroscopic device (Optrode) to measure fluorescence from SYTO 9 and propidium iodide stained cells that can be used to enumerate the bacterial load. We propose a procedure using the Optrode that will take bacteria in a clinical sample, challenge with a panel of antibiotics, and measure live/dead ratios to determine the best bactericidal choice. Using calibration data we optimised the live/dead spectrometry protocol outlined in the kit instructions, improving upon media selection for growth and staining, and analytical parameters. We applied the optimised methodology to detect live and dead Escherichia coli in populations challenged with ampicillin. Killing was detected by the Optrode in near real-time when E. coli was treated with ampicillin and stained with SYTO 9 and/or PI. Following on from the promising results generated with ampicillin, live/dead spectrometry of ampicillin challenged cells was characterised in terms of antibiotic concentration, growth phase, and susceptibility to treatment for each treatment time. The generated data demonstrated that reliable detection of E. coli knockdown by ampicillin using live/dead spectrometry requires log phase cells challenged with a suitable concentration for a particular treatment time.
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The present & the past: a review of newly accessioned bacterial strains into the UK’s National Collection of Type Cultures
More LessThe National Collection of Type Cultures (NCTC) is a bacterial culture collection which is taxonomically and biologically diverse. NCTC holds approximately 6000 different strains from over 900 different species; among them strains originally isolated in the 19th century, strains for use as controls as stipulated EUCAST and ISO guidelines, type strains on which the description of bacterial species are based and other strains from a variety of backgrounds. The remit of NCTC is to provide authentic bacterial cultures of medical and veterinary interest to the scientific community, to support and enhance the reproducibility of scientific research and to improve global public health. To fulfil this remit, remain scientifically relevant and to preserve the legacy of contemporary medical bacteriology for future scientists, NCTC accessions strains of clinical significance: such as recently circulating and outbreak strains, diagnostic escape mutants and strains with novel antimicrobial resistance profiles. In 2018, 166 bacterial strains were accessioned into the NCTC and made available to the scientific community. These include NCTC 14052: a reference strain for emergent hyper-virulent K. pneumoniae, 4 type strains of newly described bacterial species, 82 strains accessioned from the Murray Collection of pre-antibiotic era Enterobacteriaceae and 8 strains with antimicrobial resistance mechanisms previously unrepresented in the collection, including NCTC 14208: a N. gonorrhoeae isolated from an instance of combined ceftriaxone and azithromycin treatment failure. Through literature review we have highlighted their value to the scientific community, both in their own right and in the context of bacterial strains already held by the NCTC.
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Efficacy of novel eugenol tosylate congeners as antifungal compounds in combination with fluconazole against Candida albicans
More LessThe incidences of Candida albicans infections and their changing drug resistance patterns have drastically increased in recent years. Therefore, new drugs and alternative treatment strategies are promptly required. Combination therapy and the use of natural products have been extensively studied as alternative treatment. In this study, we synthesized Eugenol Tosylate Congeners (ETCs 1–6) and evaluated their antifungal activity profile alone and in combination with fluconazole (FLC) against four FLC susceptible and three FLC resistant clinical isolates of Candida albicans isolates according to CLSI guidelines. For insight mechanism of antifungal action of ETCs, activity of plasma membrane H+-ATPase pump of these C. albicans isolates was determined by monitoring the pH of the external medium. ETC 1 and ETC 4 were the most active congeners against the resistant isolates with the MIC ranging from 125 to 250 µg ml−1. The MFC of ETCs ranged from 1000 µg ml−1 to 2000 µg ml−1. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms showed 36 % of synergy, 29 % of additive, 33 % of indifferent and 2 % of antagonistic interactions. These compounds also inhibit H+efflux activity of H+-ATPase pump at varying degrees. Our results suggest that these ETCs may be directly binding to this pump and thereby inhibiting H+-efflux in Candida cells. These results advocate the potential of these compounds in developing new antifungal drugs; however, further studies are required to understand the other mechanisms involved and in vivo efficacy and toxicity of these compounds.
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Pseudomonas aeruginosa antibiogram profiles are poor indicators of genetic relatedness
More LessPseudomonas aeruginosa is a significant nosocomial pathogen responsible for severe and life threatening infections particularly in immunocompromised patients. This organism is ubiquitous in healthcare environments particularly water systems which act as a reservoir of infection. Recognition of a potential outbreak and having the ability to quickly identify and mitigate sources of exposure is critical for effective infection control. Historically analysis of P. aeruginosa antibiogram profiles represents a convenient and frequently used ‘first line’ indicator of strain relatedness. Reported here is a comparison of P. aeruginosa antibiogram profiles with those obtained using rapid Variable Number Tandem Repeat (VNTR) for patient and environmental isolates in in three separate local nosocomial outbreaks. The results demonstrate that antibiogram profiles from P. aeruginosa should not be employed as presumptive indicators of relatedness, doing so can falsely re-assure clinicians. Use of rapid molecular typing method VNTR allows genotypically identical strains to be unambiguously identified within 48 h.
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The Type III CRISPR-Cas system of Mycobacterium tuberculosis
More LessBackgroundBacteria and archaea have developed a number of strategies to keep the influx of mobile genetic elements (MGEs) such as viruses and plasmids in check. CRISPR-Cas systems provide adaptive immunity; all types of CRISPR-Cas systems have in common the genes for uptake of genetic information from the invading MGE but differ in the composition of the effector complex that directs and enforces the immune response. The genome of Mycobacterium tuberculosis encodes a Class 1, type III-A CRISPR-Cas system that has not been studied in detail.
MethodologyWe used heterologous production of the mycobacterial CRISPR-Cas genes. Purification of the enzymes enabled in vitro biochemical characterisation of the adaptation, maturation and interference proteins. Reconstitution of the system in E. coli was used to demonstrate functionality in vivo.
ResultsMaturation of crRNA through the action of Cas6 proceeded as expected, generating canonical CRISPR RNAs (crRNAs). The type III effector complex, consisting of Csm1-5, was shown to bind crRNA and cleave target RNA with the typical 6 nt spacing, display ssDNase activity and produce the cyclic oligoadenylate signalling molecule. The latter clearly activated the ribonuclease Csm6, an essential element in type III immunity. The M. tuberculosis type III CRISPR-Cas system was also reconstituted in E. coli where it provided plasmid immunity, demonstrating the functionality in vivo.
ConclusionsAll elements of the M. tuberculosis type III CRISPR-Cas system are functional in vitro and in vivo. These studies lay the foundation for further investigations into the mechanism of adaptive immunity and possible applications in biotechnology.
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Pneumococcal invasive disease preceded by intracellular replication within splenic macrophages
During bacteremic pneumonia, the prevailing dogma is that bacteria seed from the lungs into the blood. Recently, we have shown that experimental murine sepsis is preceded by intracellular replication within splenic macrophages (Ercoli Nat Microbiol 2018), which shed into the bloodstream initiating invasive disease. Here we aimed to investigate a role for the spleen in the pathogenesis of bacteraemia following pneumonia. We analysed by confocal microscopy the fate of pneumococci during ex vivo human spleen perfusions (REC reference: 18/EM/0057), in spleens during pneumonia in non-human primates (Reyes PLOS one 2016) and mice. During ex vivohumanspleenperfusion, clusters of pneumococci were observed within macrophages and the size of bacterial clusters increased over time. To associate these infectious foci to invasive pneumococcal disease during pneumonia, we analysed spleens in a baboon pneumonia model, and detected pneumococcal clusters in splenic macrophages. To test the functional relevance of these data, we treated intranasally-challenged mice with a single, non-therapeutic sub-MIC dose of azithromycin, known to concentrate inside macrophages. Data showed that bacterial lung-counts were identical in treated and untreated mice. Untreated mice showed signs of disease, had high blood and spleen-counts, whereas mice treated with the non-therapeutic dose showed no signs of disease, had low spleen-counts and no bacteraemia. Thus, the number of pneumococci in the spleen, not the lung, correlates to blood-counts during bacterial pneumonia. We hypothesise that after initial control of invasive infection by the spleen, bacteraemia associated with pneumonia arises from a sub-set of splenic macrophages that are permissive for bacterial replication.
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Clostridium difficile: cell surface biogenesis
More LessOn the C. difficile cell surface is a proteinaceous paracrystalline array, known as the S-layer. The S-layer of C. difficile is composed of two proteins: the high molecular weight (HMW) and the low molecular weight (LMW) S-layer proteins, derived from the pre-protein SlpA. PS-II, an anionic polymer found in all C. difficile strains examined to date, has been identified as the ligand responsible for the attachment of S-layer and associated cell wall proteins. Early efforts to knock out slpA proved unsuccessful and the genes thought to encode the PS-II synthesis pathway were also thought to be essential. However, by using bacteriocins that specifically target the S-layer, we recently isolated a mutant which had no evident S-layer due to a mutation in the slpA gene. As the S-layer was previously thought to be essential, it now brings into question whether PS-II is also essential. In the strain lacking an S-layer, we have now created a deletion mutant in the putative PS-II polymerase and we are attempting to generate additional mutations in the polysaccharide synthesis pathway. Analysis of these mutants will provide insights into the mechanism of PS-II synthesis and shed light on its function in cell morphogenesis.
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Nature and consequences of Salmonella infections in cattle
More LessSalmonella enterica is a human and veterinary pathogen of global importance. Cattle are a key reservoir of S. enterica serotypes that cause non-typhoidal salmonellosis in humans and infections are often acquired via consumption of contaminated food. Salmonella can survive within the bovine lymphatic system and contaminated peripheral lymph nodes often enter the food chain via ground beef production because they are small and deeply embedded in fat, making it impossible to remove them during food production. S. enterica serotypes can also cause acute enteritis and systemic typhoid-like disease in cattle, thereby exerting a significant burden on bovine welfare and productivity. Existing vaccines confer limited serotype-specific protection and a need exists to better understand the host and bacterial factors involved in pathogenesis and protection to inform the design of new vaccines and other intervention strategies. Most of our knowledge about salmonellosis comes from the mouse typhoid model. Here, we sought to understand the effects of infection by Salmonella Dublin, which causes typhoid-like disease in cattle, in its natural host. By infecting cattle with S. Dublin expressing green fluorescent protein and using flow cytometry we have been able to isolate and characterise the bovine cells infected by S. Dublin, study changes in their cell surface marker expression post-infection and compare tropism in the intestine and draining lymph nodes. We have also studied the survival of S. Dublin in the main infected host cell type in vitro using primary cells to determine the consequences of infection on the pathogen itself.
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A novel deep-sea sponge bacterium producing two promising antimicrobial candidates
More LessBackgroundNatural product screening methods are arguably the most efficient way to identify novel antibiotics. Exploiting obscure, hard to reach environments, implementing the latest high-throughput next generation sequencing techniques, performing in silico analysis and synthesis/recombinant expression of promising candidates may increase the discovery of unique agents.
Materials/methodsDeep-sea sponges were collected (∼1000 m depth) in the North Atlantic Ocean and bacteria were recovered. One strain (EU4) was selected for detailed analysis. Strain EU4 produced an inhibitor into liquid media. This compound was purified using liquid chromatography andmatrix-assisted laser desorption/ionization (MALDI) analysis was performed. In parallel, the draft genome was obtained and analysed using BAGEL-3 and anti SMASH-4.0mining tools. Successful synthetic production was obtained for a candidate identified using antiSMASH.
ResultsAnalysis ofthedraft genome (5.8Mbp) indicates that strain EU4is a novel member of the Bacillaceae. To date, the produced compound showed activity towards Micrococcus luteus only, while the synthetic compound displays a broad spectrum of activity towards Gram positive and negative bacteria. In addition, based on MALDI analysis, the synthetic and the naturally produced compounds possess different molecular weights, being approximately 4 kDa and 1.7 kDa, respectively.
ConclusionsBacteria recovered from deep-sea sponges couldpotentially be a rich source for novel compounds. In silico analysis of producer genomes has provided a means of identifying cryptic compounds, not produced in culture. Further study of both compounds, which showed diverse activity spectra, may lead to promising new candidates for development into clinically relevant therapeutics.
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The role of glutamine synthetase in the pathogenesis of Neisseria meningitidis
More LessNeisseria meningitidisis a cause of meningitis and severe sepsis. A moonlighting protein is a protein with the ability to perform additional task(s) alongside its recognised function. These proteins have been identified in both prokaryotic and eukaryotic cells and they represent a highly conserved subset of proteins that typically are either metabolic pathway-associated enzymes or act as molecular chaperones. To explore the role of GlnA in the pathogenesis of meningococcal disease, the encoding gene designated NMB0359, was amplified from the wild-type meningococcal strain MC58 and cloned into the pQE-30 expression vector. Recombinant glutamine synthetase (rGlnA) was expressed in E. coli and purified by immobilised metal affinity chromatograph. Rabbit antisera was raised against purified rGlnA (RαGlnA) and used to investigate the localisation of GlnA at the cell surface. Attempts were also made to generate glnA knockout and complemented strains of wild-type N. meningitidis. rGlnA was successfully purified from E. coli cell lysates under native conditions. A highly immuno-reactive band of the expected size (52 kDa) was observed when rGlnA immunoblot was probed with RαGlnA. GlnA could be detected on the surface of wild-type encapsulated N. meningitidis MC58 using whole-cell enzyme linked immunosorbent assay (ELISA). Surface localisation of GlnA indicates that it may be a moonlighting protein carrying out function(s) at the cell surface. Future work will investigate possible moonlighting functions which may include adhesion to host cells and proteins, regulation of the host immune response, and contribution to bacterial virulence.
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Investigating the nuclear localisation and proteolytic activity of the meningococcal App and MspA autotransporters
More LessAutotransporter proteins are major secreted virulence factors of Gram-negative bacteria. They are translocated across the inner membrane via the Sec machinery and the outer membrane via the Bam complex and a series of periplasmic chaperones, respectively. The passenger domain may then be proteolytically cleaved and released into the external milieu. The meningococcal autotransporters Adhesion and penetration protein (App) and Meningococcal serine protease A (MspA) are secreted S6-peptidase family autotransporters. Our previous work has shown that FITC-labelled recombinant App or MspA can be taken up by host cells and translocated into the nucleus. App and MspA can also bind to, and cleave recombinant host histones. We furthered investigate the ability of App and MspA (and their inactive derivatives) to cleave recombinant and host-derived histones. Our data demonstrate proteolytic activity of App and MspA on recombinant H3 and Hep-2 cell-derived H3 (which may undergo post-translational modifications that are not applied to the recombinant protein); no cleavage was observed when the histone proteins were treated with proteolytically inactive mutants of the autotransporter proteins. We have also further investigated the nuclear localisation of App and MspA by deleting areas of interest within the meningococcal autotransporters and assessing the impact on nuclear localisation in order to identify the autotransporter motifs required to direct App and MspA to the nuclear compartment. In summary, our results confirm that App and MspA can reach the nuclear compartment of the host cell and clip host-derived histone H3.
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Identification of a nitrite reductase in Pseudomonas aeruginosa as a potential antimicrobial target
More LessThe opportunistic pathogen Pseudomonas aeruginosa utilizes a wide range of virulence factors to adapt to the host environment. With the antimicrobial pipeline drying up, understanding and targeting virulence factors for therapeutic development is an exciting alternative for the discovery of novel disease inhibitors. An integrated genome-wide transposon mutagenesis screening approach was performed in P. aeruginosa using multiple in vivo disease models with the aim to identify new virulence factors required for infection. A mutant attenuated in the production of multiple virulence determinants using in vitro assays was identified. This mutant also showed severe attenuation using in vivo models with up to an 80 % increased survival in murine chronic and acute lung infection models. The predicted protein coded by the mutated gene showed homology to nitrite and sulphite reductases. Using a methyl viologen reduction assay, we have shown that this gene encondes a nitrite reductase, operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the requirement of siroheme revealed that product of this gene is an assimilatory nitrite reductase and hence we propose it to be named as NirA. Work is now on-going to understand how NirA contributes to virulence and determine the crystal structure of this protein with a view to screen for novel inhibitors of this enzyme using a drug discovery platform available in our laboratories.
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Investigation of a hospital Enterobacter cloacae NDM-1 outbreak using whole genome sequencing
Caprbapenemase-producing Gram-negative micro-organisms are emerging as a major clinical problem. The infections caused by these highly resistant and hospital-adapted pathogens may become untreatable using existing antibiotics. Over a three year period, six patients at a large UK tertiary-referral hospital were colonised or infected with carbapenem-resistant Enterobacter cloacae carrying the blaNDM-1 metallo-β-lactamase gene. Environmental isolates were also obtained from a clinical wash-hand basin and taps. The isolates had very similar pulsed-field gel electrophoresis profiles, suggesting they were related, although only four of the cases had epidemiological links. Whole genome sequencing showed the isolates had the same genomic background (sequence type ST114). Genes encoding seven different extended-spectrum and inhibitor-resistant β-lactamase and carbapenemase enzymes (blaNDM-1; blaCTX-M-15; blaACT-16; blaVEB-1; blaTEM-1; blaOXA-1 and blaOXA-10) were present, in addition to multiple genes and mutations conferring resistance to aminoglycosides, quinolones, trimethoprim, tetracycline, sulphonamide, chloramphenicol, rifampicin and fosfomycin. Phenotypic testing indicated sensitivity only to colistin and tigecycline. Genome-wide single nucleotide polymorphism analysis showed the four linked isolates were closely related, and differed from the unlinked isolates by 16–24 SNPs. Moreover, resistance encoding plasmids had been lost in the two unlinked isolates. This suggested these isolates, although sharing a recent common ancestor, had evolved in different environments. Whole genome sequencing allowed resolution of very closely related E. cloacae strains, and confirmed the outbreak did not extend beyond the linked patients. Sequencing also confirmed the same highly resistant E. cloacae strain had persisted within a clinical unit for over two years, despite rigorous efforts to eradicate it.
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Development of anti-virulence polymers targeting mycobacteria
More LessModern medicine is under the excruciating pressure of drug resistant bacterial strains which are ever advancing with the introduction of every new class of antibiotics. Traditional bactericidal and bacteriostatic drugs, while effective in eliminating the susceptible bacterial strains, also impose a selective pressure on bacteria which often leads to the emergence of antimicrobial resistance. An alternative approach is the development of anti-virulence therapies, which aims reduce bacterial pathogenesis while avoiding the selective pressure of classical antimicrobial inhibitors, thus rendering bacteria harmless and potentiating natural elimination from the host by innate immunity defence mechanisms. We have synthesised a selection of functional polymers of poly(acryloyl hydrazide) using a panel of aldehyde functionalisation groups and evaluated their anti-virulence properties on both Mycobacterium bovis BCG and Mycobacterium smegmatis mc2 155, two surrogate organisms to study Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis. Using a combination of microscopy and in vitro studies, we have shown the effectiveness of anti-virulence polymers in reducing mycobacterial phagocytosis in J774 macrophages with minimal antimicrobial activity.
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The validation of VIPcheck™ plates to screen Aspergillus fumigatus isolates for phenotypic resistance to triazole antifungal agents in St. James’s Hospital, Dublin
More LessTriazole resistance is an emerging problem in Aspergillus fumigatus (AF) resulting in failure of azole therapy. Triazole resistant AF is acquired through one of two routes – previous exposure to triazole therapy or an environmental source. In vitro antifungal susceptibility testing (AFST) on all AF strains isolated in a microbiology laboratory would be both labour intensive and impractical. A method to screen for triazole resistance would be more favourable. VIPcheck™ plates provide a simple agar based screening method. Each 4-well plate contains a growth control (GC) well and 3 wells containing itraconazole (4 mg l−1), voriconazole (2 mg l−1) and posaconazole (0.5 mg l−1). Briefly, 25 µl of a 0.5-2 McF suspension AF is inoculated into each well and plates are read after 48 h incubation at 37 °C. Any growth in a triazole containing well is suggestive of resistance. Currently in SJH, AFST is carried out using gradient strips (Liofilchem™) and results are interpreted using EUCAST breakpoints. We validated the VIPcheck™ plates with the intention to include this screening method as part of our AFST for AF isolated from clinical samples. A total of 18 isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates (n=2 wild type, n=18 resistant to ≥1 triazole drug as previously determined by AFST and/or molecular methods). The wild type isolates showed growth only in the GC well while the resistant strains all showed growth in one or more of the triazole containing wells. Our results suggest that the VIPcheck™ plate is a reliable screening method for triazole resistance.
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A SLIC answer to a continuing problem
More LessBackgroundThe burden of Anti-Microbial Resistance (AMR) is a growing problem globally. Here we present a device that determines susceptibility rapidly from primary human or animal samples and could turn the tide of AMR. SLIC (Scattered Light Integrating Collector) is a sensitive device for the detection of microbes based on the scattering of laser light.
MethodsProof of concept studies were carried out initially to establish the lower limit of detection. This was found to be 10–50 c.f.u. ml−1. This exquisite sensitivity allowed us to commence work establishing rapid MICs. Starting with an inoculum of 105/ml bacteria and using a relevant range of antibiotic concentrations the MIC can be established in less than one microbial doubling period.
ResultsThe rapid and sensitive detection SLIC affords allows for fast growing organisms such as E. coli and S. aureus to have their MICs established in less than 10 min, for any antibiotic. For slow growing organisms such as M. bovis we are able to establish an MIC in H. influenzae and Mycoplasma spp.
ConclusionAs bacterial quantification is continuously monitored we are able to see the action of antibiotics in real time. Using this facility, we can readily distinguish between lytic antibiotics and bactericidal but non-lytic antibiotics. This provides the opportunity to gain new insights into the mechanisms of action and the effect antibiotics have on microbes in a new way in a novel Point-of-Care device.
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Understanding the pathogenic process of uropathogenic Escherichia coli ST127 using proteomics on uroepithelial co-culture samples
More LessBackgroundUropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection (UTI). Strains of sequence type (ST) 127 exhibit the highest virulence potential of most UPEC strains, but little is known about pathogenicity during infection. We sought to investigate this using a quantitative proteomics approach.
Material/MethodsThree strains of UPEC ST127 (EC18, EC41 and SA189), in addition to non-pathogenic strain E. coli K12, were analysed in co-culture with the uroepithelial cell-line HT1197 for 5 h. We analyzed the bacterial and uroepithelial proteome along with the secreted proteins in the medium (secretome). The digested proteins and peptides from all fractions were separated on a Dionex Ultimate 3000 RSLC nano flow system and analyzed in an Orbitrap Velos Pro FTMS. Data were processed using Persues software.
ResultsLabel free quantitative proteomics revealed different proteomic profiles of the co-cultured strains. Gene Ontology enrichmentanalysis showed upregulation in the pentose phosphate pathway and glycolysis/glycogenesis in EC18 (an O-antigen deficient mutant). These two pathways could be important routes of carbon flux through the central metabolic pathways during growth in urine. Co-culture of SA189 with HT1197 cells leads to apparent cytotoxic effects in HT1197 cells not seen with other UPEC strains. Analysis of the SA189 secretome revealed highly abundant bacterial proteins, some of which (e.g. aromatic-amino-acid aminotransferase) were uniquely found during co-culture conditions.
ConclusionProteomics is crucial towards increasing our understanding of the pathogenic potential of UPEC ST127 strains and may facilitate identification of novel diagnostic or therapeutic targets to reduce UTI.
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Purification and characterisation of antimicrobial agents isolated from a member of the Paenibacillus genus
More LessAntimicrobial resistance (AMR) poses an ever-increasing threat to public health; the prevalence of resistant bacterial strains has reduced the clinical efficacy of many existing therapeutics and is therefore contributing to rising mortality rates due to difficult to treat bacterial infections. Two key approaches used to mitigate the threat of AMR are the discovery of novel therapeutics with activity against these resistant strains, and educating the wider public about the impact of AMR, and steps that can be taken to reduce the development of resistance. We are combining both approaches to enhance the impact of our public engagement activities. During a recent event at the University of Plymouth, a member of the public isolated the bacterial strain ‘36A’ from the button of a lift control panel. Simultaneous antagonistic screening identified antimicrobial activity against a range of both Gram-positive and Gram-negative bacteria. 36A was then subjected to draft genome sequence determination via the MinION platform (Oxford Nanopore). Growth media were optimised to enhance antimicrobial activity, with fermentation in LB broth and subsequent purification of the culture supernatant via multi-stage column chromatography resulting in the isolation of four putative antimicrobial compounds. Initial characterisation has shown that each compound has a peptidic component, all showing stability and potency at a relatively low concentration against MRSA, E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Structural characterisation has been carried out using mass spectrometry, with further characterisation and cell toxicity studies ongoing. The producing strain has been identified as a member of the Paenibacillus genus.
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Induction and characterisation of a 25-hydroxycholesterol associated immune response to Gram positive and negative bacteria in a whole blood model of sepsis
More LessEarly diagnosis and treatment of sepsis is one of the biggest challenges to ICU clinicians. Globally, 19 million cases occur annually and it is the third biggest cause of death in the UK. Sepsis is characterised by an uncontrollable, non-specific immune response to an infection, and as a result is difficult to diagnose. Recent research has found that 25-hydroxycholesterol (25-HC) plays a crucial role in the immune response to viral infection. Less is known about the role of sepsis-associated bacteria in this response. To identify novel biomarkers in bacterial sepsis a whole blood model was used and the cellular and molecular responses measured to well-characterised bacteria (Escherichia coli K12 and Staphylococcus epidermidis RP62A) using flow cytometry, ELISA and high performance liquid chromatography-mass spectrometry (LC-MS). Following bacterial infection, mononuclear cells and granulocytes decrease rapidly in response to both K12 and RP62A. This corresponds to a concomitant increase in total CD45 and CD19 expression and the concentration of the proinflammatory cytokines IL-6, CCL3 and CCL20. Proinflammatory responses were significantly more pronounced in K12 infection. There were significant increases in 25-HC in response to K12 infection, and this effect was partially blocked through inhibition of TLR2 or TLR4. Our results suggest the importance of using both cellular and humoral screening to identify unique pathways induced by sepsis causing bacteria. In addition, the current study provides some of the first evidence that 25-HC may be involved in a bacterial driven immune response. This study has importance when designing novel biomarkers to predict sepsis.
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Towards a clinically relevant model for investigation of host-microbe interactions in ventilator-associated pneumonia
More LessVentilator-associated pneumonia (VAP) is amongst the most common healthcare-associated infections worldwide. Current understanding of the underlying mechanisms has focussed on either the microbiological or physiological elements but host-microbe interactions, which are instrumental in pathogenesis, have received less research focus. This work aims to explore clinically relevant and reproducible models to investigate these interactions in the context of VAP. A clinical isolate of Pseudomonas aeruginosa, a pathogen common in VAP, was investigated in systems of increasing complexity. Sensitivity to key antibiotics (LVX, MEM, and TZP) used for treatment of VAP was unaffected by the presence of cytokines (IL-1β, IL-6, and TNFα) in vitro. Larvae of Galleria mellonella, an in vivo insect model with a rudimentary immune system, was used to test virulence of P. aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. P. aeruginosa killed 100 % of larvae within 24 h. S. aureus and K. pneumoniae killed 43.5 % and 50 % of larvae respectively, within 8 days. An ex vivo mammalian model was developed, which demonstrated abundant P. aeruginosa proliferation on lung tissue. After validating inactivation of host lung tissue, we identified changes in the expression of P. aeruginosa quorum sensing genes LasI and RhlI, specifically induced by host interaction. Our results suggest that host factors may influence bacterial growth and gene expression. We will use these early data to validate and expand our models prior to investigation of clinical samples from VAP patients. We will report our most recent findings in the development of clinically relevant models to investigate VAP.
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Development of biofilm-penetrant antimicrobial delivery system to counter Burkholderia infections
More LessMelioidosis is caused by the Gram-negative, intracellular bacterium Burkholderia pseudomallei. The epidemiology of the disease is unclear- it is limited to tropical regions, where monitoring can be sparse and further complicated by the multifaceted nature of the condition. The most common indication of acute melioidosis is pneumonia (51 %) that can progress to acute fulminant sepsis with multifocal infiltrates with high rates of mortality. Meliodosis has been estimated to cause 89 000 deaths annually with an estimated mortality rate of approximately 50 %. Due to ease of the dissemination of the pathogen, its ability to form biofilms, the high degree of antimicrobial resistance and paucity of effective treatments, controlling the infection is a major challenge. Our approach is to develop nanoparticles capable of effectively delivering antimicrobials to biofilms formed by Burkholderia species. Formulations of a proprietary lipidic delivery agent, CM2, have been tested against two species: B. cepacia UCB717 that has no capsule and B. thailandensis (that is used as a surrogate for B. pseudomallei), including strains with and without capsules both of which readily form biofilms. Confocal laser scanning microscopy was used to monitor the uptake of the nanoparticles and the MIC and MBEC of CM2 alone (and analogues), in combination with a panel of antimicrobials and a novel oligonucleotide antimicrobial, termed a Transcription Factor Decoy (TFD), were measured. The most efficacious combinations will be formulated for delivery by inhalation prior to testing in animal models.
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Acquisition of fluoroquinolone resistance in Campylobacter jejuni leads to an increase in biofilm formation and virulence
More LessCampylobacter jejuni is the leading cause of bacterial gastroenteritis with over 550 million cases reported yearly. The World Health Organization has listed C. jejuni as one of 12 microorganisms on a global priority list for antibiotic resistance due to a rapid increase in the number of strains resistant to fluoroquinolone antibiotics. This fluoroquinolone resistance is conferred through a single point mutation in the QRDR region within the gyrA gene which is also involved in DNA supercoiling homeostasis. We recently revealed that changes in DNA topology play a major role in the regulation of virulence in C. jejuni with relaxation of DNA supercoiling associated with increased attachment to and invasion of human epithelial cells. The aim of this study was to investigate whether fluoroquinolone resistant strains of C. jejuni displayed altered supercoiling associated phenotypes. A panel of mutants were derived against nalidixic acid and ciprofloxacin and shown to have a greater ability to form viable biofilms under aerobic conditions and that this phenotype was associated with changes in DNA supercoiling levels. These mutants were also shown to have an increased ability to attach to and invade epithelial cells in vitro and conferred an increase in the killing efficiency of Galleria mellonella. We report for the first time that fluoroquinolone resistance in C. jejuni is associated with an increase in virulence and the ability to form viable biofilms in oxygen rich environments. These altered phenotypes may play a critical role in the continued increase in fluoroquinolone resistance observed for this important pathogen.
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Investigating the uptake mechanism of S type pyocins
More LessPseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen with a high mortality and morbidity rate. It has many mechanisms of resistance to antibiotics, which makes it hard to treat. Pyocin is a protein that is produced by P.aeruginosa that kills related strain bacteria. pyocin type S bind to ferrisiderophore receptors that uses the TonB system to translocate into the bacterial cell. The aim of this study was to express and purify receptor and translocation (R+T) domains of pyocin S1, S2 and S3 plus the TonB1 receptor and ToLAIII receptor of P.aeruginosa to determine if pyocins S uses these to transverse the inner membrane of target cells. IPTG was used to induce the protein in the expression and analyzed by SDS-PAGE gel, giving fragment size of 11 kDa (ToLAIII), 23 kDa (TonB1), 45 kDa (S2 R+T) and 79 kDa (S3 R+T). Protein purification was cried on the four proteins using affinity chromatography technique by His-tag in C-terminal of S2 and S3 R+T domain, N-terminal of TonB1 and ToLAIII proteins. In addition, Gel Filtration chromatography was used to further purify the proteins so that the interaction between them could be tested. This study confirmed that the proteins used is expressed and purified, so in the future study the gel filtration would be carried out for R+T domains of the other S pyocins and TonB1 protein so that the interaction would be tested between the proteins in the study.
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Gut microbiota derived mitochondrial inhibitors cross the blood brain barrier and localise white matter
The microbiome-gut-brain (MGB) axis is a bi-directional route of communication that exists between the brain and the microbes that reside in the gut. The MGB axis is becoming of increasing importance as significant alterations in the gut microbiota are now linked to numerous neurological conditions, however, little is currently known about the microbiome derived mediators of communication. Here we used mass spectrometry imaging (MSI), a label free imaging technique, to identify bacterial products that cross the blood brain barrier in specific pathogen free (SPF) mice. We identified two bacterial molecules abundant in white matter regions of the murine that were absent in the brain and gut in germ free (GF) mice. We have identified the primary gut microbial producers of these metabolites to be members of the Lachnospiraceae family. Both molecules were found to be structurally similar to carnitine and localise with carnitine in the SPF mouse brain. Using a primary murine cell culture model of the central nervous system white matter we show that these molecules are capable of significantly impairing mitochondrial basal respiration. Given their systemic presence in the mouse and their presence in human biological samples, these metabolites may have significant implications for diseases associated with mitochondrial dysfunction and an altered gut microbiota. These results are the first to describe a direct molecular inter-kingdom communication between prokaryotes and the mammalian brain that can facilitate functional inhibition in mammalian brain cells.
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The UK public health rapid support team: a novel programme integrating outbreak response, operational research, and capacity building
The 2013–16 epidemic of Ebola virus disease underscored the shortcomings of the international community to both respond to outbreaks and conduct critical research in complex humanitarian crises. To address these concerns, the UK Government has formed the UK Public Health Rapid Support Team (UK- PHRST). The UK-PHRST is a collaboration between Public Health England and the London School of Hygiene and Tropical Medicine with the University of Oxford and Kings College London as academic partners. The UK-PHRST has a novel triple mandate to work in low- and middle-income countries (LMICs) to:
- Respond to outbreaks
- Conduct innovative operational research during and between outbreaks to generate evidence on best practices
- Build LMIC and regional capacity for outbreak response
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Functionalised liposomal formulations for delivery of antibiotic agents
Antimicrobial resistance is a major global healthcare challenge. Beyond the discovery of novel antimicrobial agents, the development of novel formulations for enhancing current antibiotics is a promising strategy to reduce the rate of treatment failure. Drug delivery nano-carriers can achieve high local concentration of antimicrobial agents, reduce toxicity, and improve biodistribution and pharmacokinetics. We have taken two approaches to enhance antibiotic delivery and effectiveness. Firstly, we used a bespoke targeted liposomal system for intracellular antibiotic delivery to phagocytic cells. This enables treatment of an intracellular Gram-negative infection with a cell-impermeable antibiotic. Targeted liposomes were found to significantly enhance uptake compared to uncoated liposome control formulations both in in vitro and in vivo (zebrafish model). Secondly, liposomal nanoformulations were utilised to deliver peptide antibiotics, where liposomes protect peptides from degradation, and allow potential co-delivery of combination therapeutics. We investigated different liposomal formulations and drug combinations against E. coli and S. aureus. We show that peptide-loaded liposomes are more efficient compared to free drug in inhibiting the growth of both Gram-negative and Gram-positive bacteria. These initial results suggest that liposome-mediated delivery can be utilised for the repositioning and repurposing of existing antibiotics, potentially allowing for the treatment of diverse infections in a more effective manner.
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Continuous culture of Escherichia coli, under selective pressure by a novel antimicrobial complex, does not result in the development of resistance
More LessAntibiotic resistance is a major global health problem. Preservation of antibiotics, underpinned by the availability of novel antimicrobials that avoid the emergence of antimicrobial resistance and antibiotic cross-resistance is an important societal goal. We have developed a novel biocidal complex (iodo-thiocyanate complex or ITC), drawing the inspiration from naturally occurring peroxidase-catalysed systems. This study was aimed to reveal the potential of ITC for induction of resistance and cross-resistance, and, thus, different aspects of resistance were explored. We show that the repeated exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus to sub-inhibitory levels of ITC during serial passage of batch cultures did not generate ITC resistance. By comparison, E. coli and S. aureus developed low-level and high-level resistance to levofloxacin (LVX), respectively. Further, we attempted to generate de novo resistance in antimicrobial-sensitive E. coli during 20 days of continuous culturing when exposed to gradually increasing concentrations of ITC and LVX. The exposure of E. coli to ITC did not induce resistance to ITC, or cross-resistance to LVX. No distinct mutational pattern was evidenced from whole-genome sequence (WGS)-based analysis of ITC-challenged bacterial populations. By contrast, the resistance to LVX was rapidly induced, selected for high-level and enriched with a distinctly characteristic genome mutational pattern. WGS of LVX-challenged population revealed that the majority of mutations appeared in the genes of LVX target proteins and drug influx. This study suggests that the usage of ITC may not trigger the emergence of facile resistance or cross-resistance, in contrast to common antibiotics.
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Do Streptococcus pneumoniae and respiratory Syncytial virus synergise to promote invasive disease?
More LessStreptococcus pneumoniae (S.p.) and Respiratory Syncytial Virus (RSV) are two major pathogens commonly found to coexist in respiratory secretions in patients with acute respiratory infection. Though there is increasing evidence of a synergistic interplay between these two pathobionts, the exact mechanisms remain obscure. The aim of our study was to decipher how coinfection with RSV alter pneumococcal growth dynamics and host immune response and how this impact on the colonisation and invasive properties of S.p. Using in vivo mouse model, we made the key observation that upon coinfection with RSV, the density of pneumococcal colonisation in the nasopharynx and dissemination to the lower respiratory tract were significantly higher in mice previously colonised with S.p. These mice also presented more severe weight loss and delayed recovery compared to mono-infected animals as well as significantly heightened pro-inflammatory cytokine profiles. Measurement of in vitro transepithelial electrical resistance (TEER) showed that, upon RSV coinfection, S.p. transmigrate through the epithelial barrier without altering epithelial integrity suggesting a transcellular mechanism rather than paracellular migration. Moreover, RSV-pneumococcal coinfection of human primary nasal epithelial cell demonstrated major changes in host protein expression involved in the catalytic activity, ubiquitination, cytoskeletal organisation, and endocytosis. Simultaneously, significant upregulation observed in bacterial proteins involved in the ribosomal activity, streptococcus-induced tissue inflammation, DNA supercoiling, and bacterial viability during oxidative stress, affecting both the survival and the virulence of S.p. Our results explain the complex interactions between pneumococci, RSV and host and help towards further understanding the significance of viral-bacterial co-infection in clinical settings.
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Effect of metronidazole on microbiomes associated with asymptomatic bacterial vaginosis
Asymptomatic Bacterial Vaginosis (BV) damages vaginal epithelium increasing risk of sexually-transmitted infections. Gardnerella vaginalis, Atopobium vaginae, Prevotella amnii, P. bivia and Candida albicans are associated with BV. Presence of Lactobacillus spp. is indicative of a healthy microbiota. Symptomatic BV is treated with metronidazole. The role of microbiota and metronidazole treatment in the recurrence and persistence of asymptomatic BV remains to be elucidated. This study uses whole genome sequencing (WGS) to determine the microbiota changes with metronidazole treatment. DNA from 20 vaginal swabs was obtained at four time points over 12 months from five African American women and was subjected to WGS. The first time point is the untreated baseline. All subjects were tested every 4 months and received a course of metronidazole for each episode of BV during the 12 months period. Nugent scores were used to classify BV status. The microbial profiles were analyzed along with the sociodemographic metadata. Despite treatment, the participants did not recover from BV — two participants experienced persistent BV, and the rest had recurrent BV. WGS analyses show that G. vaginalis was the most abundant organism as compared to Lactobacillus species. The metronidazole treatment resulted in the loss of Lactobacillus and Prevotella species. One participant scored healthy based on Nugent score at one time point, during when Lactobacillus species dominated the microbiome. Based on this pilot longitudinal study, metronidazole may not be an effective treatment for asymptomatic BV. Studies with larger cohorts can lead to statistically significant conclusions to develop alternative interventions for asymptomatic BV.
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Development of ‘smart’ wound dressings for biofilm sensing and control
More LessChronic wounds affect approximately 2 % of the worldwide population and incur healthcare costs in the billions. Key to their persistence is the formation of microbial biofilms, which are accounted for in nearly 80 % of all non-healing wounds. The smart dressing presented herein aims to detect a range of volatile infection protagonists, with a striking colour change that can be visualised with the naked eye, providing 24/7, non-invasive monitoring of infection development and antimicrobial treatment efficacy. A range of coloured indicator films housing dyes responsive to volatile analytes in the wound headspace were developed and tested against porcine skin inoculated with Pseudomonas aeruginosa. Digital images of the indicator film were captured at regular time intervals and the resulting images were aligned and split into red, green and blue (RGB) colour channels to yield semi-quantitative data. vAPCI-MS was exploited to identify additional volatiles for incorporation into the smart dressing design. A CO2-sensing film comprising xylenol blue dye underwent a marked colour change from blue to yellow within 12 h of inoculation with PAO1, whilst indicators monitoring uninoculated control skin remained blue (no colour change). In addition, vAPCI-MS identified putrescine as an additional volatile of interest, and responsive indicator films were developed for its detection. The marked colour change exhibited by each indicator film is easily visualised by eye and can be digitally analysed to provide semi-quantitative data. This early warning, point-of-care technology is a promising candidate in combatting biofilm development in wounds.
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Advanced titanium dioxide-polytetrafluorethylene (TiO2-PTFE) nanocomposite coatings on stainless steel surfaces exhibit significant antibacterial and anti-corrosion properties
More LessBacterial infection and corrosion are the two of the most significant causes of metallic implant failure. In our study, we innovated a facile two-step approach to synthesising a TiO2-PTFE nanocomposite coating on stainless steel, which endows the implant surface with both antibacterial and anticorrosion properties. By harnessing the adhesion and reactivity of bioinspired polydopamine, the TiO2-PTFE coating was uniformly deposited onto substrates by using a sol-gel dip coating technique. The TiO2-PTFE coating exhibited minimal bacterial adhesion against both Gram-negative Escherichia coli WT F1693 and Gram-positive Staphylococcus auerus F1557. Moreover, it was observed that an increasing TiO2 concentration in the bath enhanced antibacterial activity. Benefiting from the synergistic effect between TiO2 and PTFE, the TiO2-PTFE coating showed improved corrosion resistance in artificial body fluids comparing with the sole TiO2 and PTFE coatings. The TiO2-PTFE coating also demonstrated extraordinary biocompatibility with fibroblast cells in culture, making it a prospective useful strategy to overcome current challenges in the use of metallic implants.
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Old drugs learn new tricks – repurposing phenothiazines to uncover effective antimicrobial
More LessThioridazine (TZ) is an antipsychotic drug that acts against antibiotic resistant bacteria. The main aim of this study was to uncover the mechanism of action of TZ using Salmonella enterica serovar Thypimurium as a model bacterium. The antibacterial activity of TZ was initially determined based on its minimum inhibitory concentration (MIC). Membrane permeability assays were performed and fluorescence measured using the Ethidium Bromide accumulation assay. Salmonella was exposed to TZ and its effects on membrane potential and cell wall assessed by flow cytometry and Transmission Electron Microscopy, respectively. Effects on the bacterial proteome were assessed through 2D gel electrophoresis. Infection assasys were performed in THP-1 ad RAW 264.7 cells treated and non-treated with TZ. The MIC of TZ against Salmonella was 200 mg l−1. Our in vitro data demonstrates that TZ mechanism(s) of action involves primarily Salmonella’s membrane by affecting its permeability and potential after 15 min of exposure to TZ. At half of the MIC, and only after 15 min, TZ disrupts the bacterial membrane leading to leakage of the cellular contents and lysis of Salmonella. Proteomic profiling revealed 75 upregulated and 62 downreuglated proteins. Infected macrophages treated with sub-MIC of TZ, showed a reduction on intracellular c.f.u./mL. This may be indicative of TZ’s ability to enhance the killing activity of infected macrophages. The results obtained suggest that TZ may act in vitro by targeting the bacterial cell-envelope. Due to its effect on infected macrophages, TZ may be considered a useful adjuvant to current therapeutics.
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