- 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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