- Volume 4, Issue 5, 2022
Volume 4, Issue 5, 2022
- Abstracts from Annual Conference 2021
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- Poster Presentations
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Comparison of the Antimicrobial Efficacy and Germicidal Efficiency of 405-nm Light for Surface Decontamination
More LessBackgroundThe persistence of infectious organisms on hospital surfaces presents a significant challenge to healthcare environments. Low irradiance visible 405-nm light has recently been developed as a method for environmental decontamination, with studies demonstrating successful reductions of environmental bacteria in wards and operating theatres. This study investigates the antimicrobial efficacy of 405-nm light for decontamination of surfaces, and how the dose-response kinetics are affected by use of differing light irradiances.
MethodsSurface-seeded Staphylococcus aureus and Pseudomonas aeruginosa (selected as model Gram-positive and Gram-negative species) were exposed to increasing doses of 405-nm light (≤ 90 Jcm-2) at three discrete irradiances (0.5, 5 and 50 mWcm-2). For both species, inactivation kinetics at each respective irradiance was established and susceptibility at equivalent light doses compared.
ResultsResults demonstrate increased bacterial susceptibility to 405-nm light inactivation when exposed at lower irradiance treatments. For both species, 3 Jcm-2 was required when exposed using 0.5 mWcm-2 irradiance to achieve significant bacterial inactivation (P < 0.05; 26.7-73.7% reduction). When exposed at 5 mWcm-2, double the energy (6 Jcm-2) was required to achieve similar reductions. Exposure at the highest irradiance (50 mWcm-2) required 3-5 times greater dose (9-15 Jcm-2) to achieve similar reductions to the lowest irradiance tested (0.5 mWcm-2).
ConclusionThis study provides evidence of the enhanced germicidal efficiency of low irradiance 405-nm light, highlighting its efficacy for continuous environmental decontamination applications. Further investigation into the photo-chemical inactivation mechanisms will be crucial for its optimisation for a range of infection control applications.
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A Legionella’s outbreak in a COVID-19 pandemic setting
More LessIn this report, the author intends to reinforce the need to test for another potencial causes of pneumonia which present with similar symptoms of SARS-CoV-2 infection and can also be responsible for outbreaks and sometimes fatal, particularly Legionnaires’ disease.
Legionellabacteria are aerobic, gram-negative, ubiquitous freshwater and soil inhabitants. Pneumonia is the most commonly described manifestation of Legionella infection (Legionnaires’ disease), acquired through inhalation of aerosolized water droplets containing the bacteria.
Legionella pneumophilais the most consistently reported species, being an important cause of severe community-acquired pneumonia that often requires hospitalization and is fatal in approximately 10% of cases. Most cases present sporadically, however, outbreaks can occur when there are appropriate conditions for bacteria to grow and spread, like stagnating water in piping systems in large facilities. Furthermore, the pathogenesis of Legionella pneumonia is complex and it is clinically and radiographically similar to other forms of pneumonia.
In the context of pandemic COVID-19, the use of Legionella urinary antigen testing continues to be crucial and strongly recommended. Thus, in the outbreak faced in the author’s hospital, this screening method proved to be effective and all cases of Legionellapneumonia were promptly recognized and notified, allowing public health measures to be taken to prevent the development of other potencial clusters.
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K1 Escherichia coli form foci in the brain at 24h post infection
More LessK1 capsule type Escherichia coli is the predominant cause of neonatal meningitis. In an intravenous (IV) infection model with a clinical K1 isolate IHE 3034 adult mice die of sepsis within 24 hours. However, by utilising cefazolin, a first-generation cephalosporin known for its inability to pass through the blood brain barrier, it is possible to prevent bacteraemia and study the early phases of E. coli meningitis in the brain. Mice were infected IV with E. coli IHE 3034 and treated with cefazolin at 12 and 24h intraperitoneal to ensure the survival of animals to the determined end points. The results of the experiment show IHE 3034 is present in the brain as early as 12h and it remains in the tissue 72h post infection even if blood is clear. Despite this, at the cellular level, clear foci formation can be observed in the meninges and the choroid plexus around the vascular endothelial cells at 48h, showing IHE 3034’s ability for intracellular replication inside the meningeal region The foci do not appear to be present at 24 or 72h, indicating for a secondary replication site around the major vascular endothelia sites of the brain. This further promotes the bacterial load increase through cell bursting in the tissue with or without a sustained bacterial influx from the blood. These findings show that K1 E. coli can form foci in the brain which may lead to escalated disease progression and severe morbidity following an initially successful clearance of bloodborne bacteria during sepsis.
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Bioinformatic Analysis for Iron-Uptake System of Campylobacter spp. in humans and chickens
More LessAsymptomatic Campylobacter jejuni colonisation is highly prevalent in chickens, however, it’s a major cause of bacterial foodborne disease in humans. Iron is an essential co-factor in many physiological processes and Campylobacter strains employ several non-redundant iron acquisition systems for survival and colonization. The outer membrane receptors CfrA and CfrB are involved in iron acquisition fromcatecholamine siderophores and are required for colonisation of the chicken GI tract. In contrast, ChuAB are part of an uptake system, enabling the utilization of iron in haem and is not required for colonisation. This study aims to compare differences in the iron-uptake systems present in Campylobacter strains isolated from chickens or humans.
Analysis of Campylobacter jejuni/coli genome sequences in PubMLST (https://pubmlst.org/organisms/campylobacter-jejunicoli [https://pubmlst.org/organisms/campylobacter-jejunicoli]) added from the UK in 2018 reveals differences in sequence type (ST) and chuA, chuB, cfrA and cfrB between isolates from chicken (CI) or human (HI) sources. ST828 was the most common ST for the C. coliCI (45%) and ST827 (32%) for the HI. C. jejuniST5136 was the most common of the HI (10%) and CI (13%), ST50 (8%) for the HI and ST21 (7%) for the CI. >95% of isolates contained chuAand chuB, we also found the Chu system to be highly conserved. For cfrA81% of HI and 74% of CI contained the gene, and 92% of HI and 96% of CI harboured cfrB.<3% of all isolates had neither cfrAor cfrB, cfrBmore associated with CI than HI. This data analysis reveals potential gene targets towards vaccine development against Campylobacter strains.
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Detection of Extended Spectrum Beta-lactamase Gene (CTX-M) among Representative Multidrug-Resistant Gram Negative Bacterial Isolates from Patients with Urinary Tract Infection in Ekiti State, Nigeria
More LessUrinary tract infection is huge public health burden and the emergence of extended spectrum beta lactamase producing bacterial pathogens increases the burden of infectious diseases in Nigeria. This study determined the current prevalence of cephalosporin resistance among Gram-negative bacteria isolated from patients with urinary tract infections between February 2018 and June 2018. Non-repetitive Gram–negative bacteria were recovered from 106 individuals with urinary tract infections who reported at two tertiary healthcare centers in Ekiti-State, Nigeria. A total of 106 bacterial isolates were obtained which included: Klebsiella pneumoniae 34 (29.1%), Klebsiella oxytoca 17 (16.0%), Proteus vulgaris 10 (9.4%), E. coli 24 (22.6%), Proteus mirabilis 18 (16.9%) and Pseudomonas aeruginosa 3 (2.8%). Sixty five of these organisms showed resistance to ceftazidime while 76 organisms showed resistance to ceftriaxone. Forty representative organisms were selected and tested for presence of extended spectrum beta-lactamase (ESBL) genes using primers specific for different ESBL genes. A total of eight (20.0%) organisms carried the blaCTX-M gene and other variants of the ESBL genes were not detected. The organisms carrying the blaCTX-M gene included E. coli 3 (37.5%), K. pneumoniae 1(12.5%), P. mirabilis 1(12.5%),) and K. oxytoca 3(37.5%). The high prevalence of cephalosporin resistant Gram-negative bacteria among patients with UTI is a serious threat to public health and efforts must be intensified to regulate the clinical use of the cephalosporins.
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Profiling the effects of acne therapeutics, including the novel antibiotic sarecycline, on the human microbiota
Many factors shape the human intestinal microbiota, some of which can confer a deleterious effect on the microbiota, e.g. antibiotic therapy. Disruption to the microbiota has been implicated in the progression of C. difficile infection (CDI), multiplication of multi-drug resistant organisms and many extra-intestinal diseases. Thus, determining the off-target effects of antibiotics is essential to determine a patient’s risk of these diseases, particularly for new therapies. Here we characterise the effect of sarecycline, a novel tetracycline antibiotic for the treatment of moderate to severe acne vulgaris; and compared its effect to the gut microbiota with other acne treatments. Using four independent in vitro gut models, we exposed the human microbiota to either sarecycline, minocycline, doxycycline, or clindamycin, and monitored the changes to the bacterial populations, and whether these changes were sufficient to induce CDI.
Sarecycline or doxycycline exposure caused a temporary reduction in the bacterial diversity upon initial exposure. Sarecycline exposure was characterised by a transient increase in Enterococcus spp. and Enterobacteriaceae, and a decrease in Bifidobacterium spp. Doxycycline exposure caused longer-term changes to the Lactobacillaceae and Ruminococcaceae populations. Minocycline exposure resulted in a dramatic reduction to the bacterial diversity, with extensive expansions to the Enterococcus spp. and Enterobacteriaceae populations, whilst the Lactobacillaceae and Ruminococcaceae populations contracted. Whilst clindamycin did induce simulated CDI, neither sarecycline, minocycline, nor doxycycline created a niche conducive for CDI.
These data show that long-term sarecycline use has a lower potential for disruption of the colonic microbiota, compared with the current treatments for acne vulgaris.
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Cell-to-cell ATP differences can modulate cellular decision-making
More LessCells generate phenotypic diversity both during development and in response to stressful and changing environments, aiding survival. Functionally vital cell fate decisions from a range of phenotypic choices are made by regulatory networks, the dynamics of which rely on gene expression and hence depend on the cellular energy budget (and particularly ATP levels). However, despite pronounced cell-to-cell ATP differences observed across biological systems, the influence of energy availability on regulatory network dynamics is often overlooked as a cellular decision-making modulator, limiting our knowledge of how energy budgets affect cell behaviour. Here, we consider a mathematical model of a highly generalisable, ATP-dependent, decision-making regulatory network, and show that cell-to-cell ATP variability changes the sets of decisions a cell can make. Our model shows that increasing intracellular energy levels can increase the number of supported stable phenotypes, corresponding to increased decision-making capacity. Model cells with sub-threshold intracellular energy are limited to a singular phenotype, forcing the adoption of a specific cell fate. We suggest that energetic differences between cells may be an important consideration to help explain observed variability in cellular decision-making across a broad range of biological systems, including bacteria and the blood stem cell system.
*NOTE: Our work is highly interdisciplinary and if you believe it would be better suited to another topic area, then I would be delighted to discuss that further.
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Shiga toxin-producing E. coli (STEC) isolated from wild mammals in Portugal
More LessBackgroundZoonoses are diseases common to humans and animals (livestock, wildlife, and pets). In 2018 about 360 000 zoonoses were reported in European Union. Shiga toxin-producing Escherichia coli (STEC) infections were among the most reported causes of these zoonotic diseases.
MethodsFaecal samples of mammal species (n=286) with distinct phenology (wild boar, red deer, otter, and red fox) were collected in Portugal. After the initial processing, the presence of STEC was screened by PCR, and suspicious samples were platted on CHROMagar STEC. STEC positive isolates were tested for antibiotic susceptibility. Thephylogenetic relationship of STEC strains was evaluated by PFGE. Of these, 20 representative strains were selected for whole genome sequencing with the Illumina NovaSeq 6000 system. For the assembly, annotation and genome characterization, multiple web-based bioinformatic tools were employed.
ResultsCultivable STEC (n=52) were recovered from 17% (n=49) of the samples collected from the four mammals. All the isolates were non-O157:H7 STEC encoding stx1 (n=2; 4%) and/or stx2 genes (n=51; 98%). Only one strain (2%) of red fox was resistant to ceftazidime, aztreonam and nalidixic acid. The 20 strains that were sequenced belong mainly to serotype O27:H30 (n=15), followed by O146:H28 (n=2), O146:H21 (n=1), O178:H19 (n=1) and O103:H2 (n=1). In addition to stx, all strains encode several virulence factors, mainly toxins, adhesins, fimbrae, secretion systems, among others. Additionally, several pathogenicity islands have been predicted for these strains.
ConclusionsOur results show that wild animals are reservoirs of STEC, potentially pathogenic to humans.
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Ruminococcus gnavus GH98 substrate specificity to blood group A antigen contributes to mucin glycan foraging
The human gut symbiont Ruminococcus gnavus displays a strain-specific repertoire of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity networks showed that R. gnavus GH98 (RgGH98) sequence fell in a cluster different from sequences of GH98 enzymes functionally characterised to date. We heterologously expressed and purified RgGH98, and determined its substrate and linkage specificity. We showed that RgGH98 is specific for blood group A antigen (BgA), as also confirmed by isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR, revealing affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 specificity was further investigated using a combination of site-directed mutagenesis and X-ray crystallography. The crystal structure of the complex between RgGH98 and BgA trisaccharide and RgGH98 inactive mutant with BgA tetrasaccharide identified residues involved in RgGH98 unique specificity. RNAseq and qPCR analysis showed that the gene encoding RgGH98 is part of an operon that is overexpresssed in vitro when R. gnavusis grown on mucin as sole carbon source. We showed that RgGH98 releases BgA trisaccharide from mucin and that pretreatment of mucin with RgGH98 conferred other R. gnavusstrains lacking this enzyme the ability to grow through BgA metabolism and access to the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enables them to colonise different nutritional niches in the gut and provide a source of enzymes with unique specificities for potential applications in diagnostic or therapeutics.
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Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Despite on-going vaccination programmes, Neisseria meningitidisis a majorcause of septicaemia and meningitis. In 2017-18, the MenW and MenY capsular groups caused 38% of all UK invasive meningococcal disease cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to determine how genetic variation contributes to phenotypic differences between carriage and disease isolates. We have adapted assays, mimicking carriage and disease behaviours, for high-throughput phenotypic testing of 335 MenW cc11 and MenY cc23 isolates. We are currently testing MenW cc11 disease and carriage isolates for cytotoxicity in a human lung epithelial cell line, growth in media and biofilm formation. Phenotypic differences are utilised as inputs for Genome Wide Association Studies enabling linkage of specific genomic variants, or variant combinations, with phenotypic variation. Genomic data include whole genome sequences and repeat-mediated phase variation states.
The MenW cc11 isolates span two known phylogenetic clusters, original and 2013. Our preliminary data from high-throughput growth and biofilm assays showed no significant differences between these groups or sources (disease versus carriage); however, variations were observed within groups with, for example, distinctive cytotoxicity or biofilm differences between isolates. These variations may reflect physiological divergence due to minor genetic modifications between highly phylogenetically-related strains. Our assay systems are robust, reproducible, and easily scalable for efficient high-throughput genotypic and phenotypic testing. Thus, large-scale screening of phenotypic variation for infectious diseases is achievable and harnessable for cost-effective, direct evolutionary and epidemiological studies.
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Controlling the lytic switch; can m6A-modified RNA be used as an Anti-Viral Target?
KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019).
Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner.
Generating in silico 3D structures of the ORF50-1 RNA in its native ‘N’ and m6A-modified ‘M’ form confirms that the presence of the m6A-modification has repercussions in the lower part of the stem-loop and predicts a different secondary structure(collab. Pasquale and Roeder).
Due to the obvious structural differences anticipated, RNA-binding ligands have been identified using Small Molecule Microarrays (SMMS) (collab. Fullenkamp and Schneekloth). By investigating these ligands in biophysicalexperiments, we have verified RNA-binding and optimised cell-based assays to assess anti-viral properties. It is hoped these experiments will highlight the importance of A versus m6A within the lytic phase of KSHV’s lifecycle.
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Genetic manipulation of Lactobacillus to produce optically pure lactic acid following fermentation using hydrolysed organic fraction of municipal solid waste as a feedstock
More LessBackgroundLactic acid for polymer applications must be optically pure to allow the final plastic properties to be controlled. We have isolated a strain of Lactobacillus plantarum capable of utilising a range of carbohydrates and tolerant to high sugar and organic acid concentrations. However, like most Lactobacillus plantarum, this strain produces D- and L-lactic acid. To improve the commercial application of this strain gene editing has been used to ensure only one isoform is produced.
MethodsUsing two-step homologous recombination, ldhL was replaced with a truncated version interrupted by a chloramphenicol acetyltransferase resistance marker, then with an unmarked truncated version.
The optical purity shift was quantified biochemically by enzymatic assay and HPLC and fermentation performance compared against the unmodified strain using enzymatically hydrolysed municipal solid waste pulp (MSW sugars) as a feedstock.
ResultsThe ldhL gene was successfully deleted, leading to a dramatic shift in the optical purity of lactate produced in both synthetic media and MSW sugars. The unmodified strain produced D-lactic acid with an optical purity of 40%, while the mutant achieved 94%. In MSW sugars, the modified strain performed as well as the unmodified control, consuming all available glucose in less than 48 hours with a volumetric productivity of 1.02 g/L/h.
ConclusionsDeletion of ldhL from our strain of Lactobacillus plantarum allowed exclusive production of D-lactic acid from MSW sugars. This provides a laboratory scale demonstration of the production of optically pure lactic acid from renewable sugars for use in the production of biodegradable plastics.
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Palmatine and berberine chloride synergistically Inhibit NanH sialidase of Tannerella forsythia
More LessThe periodontal pathogen Tannerella forsythia is associated with severe periodontitis, and expresses NanH sialidases that cleave sialic acids by hydrolyzing the glycosidic bonds to underlying sugars. Palmatine and berberine chloride are plant-derived alkaloids with pharmacological effects, including anti-inflammatory and anti-bacterial properties. Recombinant NanH sialidase was purified using HisTag affinity chromatography while sialidase activity was determined using 4-methylumbelliferyl N-acetyl-α-D-neuraminic acid (MUNANA) as a substrate. The individual and synergistic effects of palmatine and berberine chloride on NanH sialidase inhibition was determined as well as their antimicrobial effects. The IC50 values of palmatine and berberine chloride were found to be 0.143 and 0.474 mM respectively. A significant synergistic effect was observed when a 0.20 mM:0.50 mM Palmatine:Berberine chloride mixture was used, inhibiting NanH sialidase by almost 100%, as compared to 0.2 mM palmatine and 0.5 mM berberine chloride invidually, which inhibit sialidase activityby 60.33 and 55.94%, respectively. Additionally, an antimicrobial viability assay was conducted and, 0.5 mM palmatine and 0.45 mM berberine showed a significant antimicrobial activity against Tannerella forsythia. Lastly, to examine potential toxicity to host cells, thecytotoxic effects of palmatine (0.15 mM) on H357 oral squamous carcinoma cells was investigated using a trypan blue assay and palmatine was found not to be toxic. In summary, a combination of palmatine and berberine display significant synergistic inhibitory effects on NanH with minimal cytotoxic effects as well as potential antimicrobialeffects on the oral pathogen T. forsythia. Suggesting thatthese compounds may have potential for future development.
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Covariation analysis of the master bacterial DNA replication initiator DnaA
More LessBacterial DNA replication initiation is governed by DnaA: an oligomeric protein belonging to the ATPases associated with diverse cellular activities (AAA+) superfamily. The master replication initiator is present in all known bacterial pathogens; thus, it is an attractive target for drug discovery. Here, an in-silico evolutionary covariation analysis of DnaA amino acid residues was conducted using EVcouplings, a Python framework, to investigate connections between key protein residues. Several co-evolving amino acid pairs were identified and characterized using PyMol to visualize the residues on DnaA crystal structures. I hypothesize that some co-evolving pairs, particularly those spatially connected, are likely important for structural integrity of the protein. Interestingly, one co-evolving pair of residues (A. aeolicus Val121 and Ser229) was found to be separated by ~27 angstroms, suggesting a more complex relationship connects these two sites. Thus, analysing co-evolution of DnaA residues supports known structural information and may predict either allosteric connections within DnaA or contacts between DnaA and other binding partners.
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Global diversity and potential functioning of prophages in plant pathogenic Ralstonia solanacearum bacterium
More LessRalstonia solanacearum is a destructive plant pathogenic bacterium which harbours a wide variety of virulence genes allowing it to infect over 200 plant species worldwide. Its virulence is also affected by the presence of integrated bacteriophages, termed prophages. While several such prophages have been identified, the global distribution and diversity of R. solanacearum prophages is unknown.
To study this, we first identified prophages present in a diverse collection of 192 assembled R. solanacearum genomes. Prophage diversity was explored by calculating prophage genetic distances and clustering with characterised prophages in a neighbour-joining tree. Prophage clusters were further verified by assessing gene content, GC content, and prophage length. Prophage identities were determined using the NCBI Virus database, and prophage-encoded virulence genes identified by analysing pangenome content.
343 intact prophages were identified, forming ten prophage clusters with distinct gene content, GC content, and length profiles. Five prophage clusters, containing 159 prophages, belonged to the Inoviridae, Myoviridae, and Siphoviridae phage families. The remaining 184 prophages were uncharacterised and may therefore represent novel prophages. Transcriptional regulators with potential virulence effects were identified in three prophage clusters, including one uncharacterised cluster. These prophage clusters were unequally distributed throughout the R. solanacearum population being host genotype specific.
This research demonstrates that R. solanacearum contains a high level of uncharacterised prophage diversity and highlights novel prophages that could contribute to pathogen virulence. Given their potential host-genotype-specific virulence effects, R. solanacearumprophages could be co-evolving with their hosts, and may contribute to global variation in R. solanacearum virulence.
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Bacteriological and Physicochemical Assessment of Sachet and Bottled Drinking Water Sold in Makurdi Metropolis, Benue State, Nigeria
More LessBackgroundPackaged drinking water popularly known as “sachet and bottled water” serves a large percentage in increasing access to clean drinking water in Nigeria. But little attention is given to investigate the bacteriological assessment of these water which may be harmful for human consumption.
ObjectiveThis study was to assess the bacteriological and physicochemical characteristics of sachet and bottled water sold in Makurdi metropolis.
MethodologyA cross sectional study was carried out with a total of one hundred and sixty-five samples collected. These comprised of triplicates of fifty sachet water and five bottled water brands purchased using simple random sampling from street vendors within Makurdi metropolis and analyzed using standard methods and results were compared with the recommended guidelines for water quality.
ResultsThe presence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., Klebsiella sp., Salmonella sp. and Shigella sp. were detected in the water samples with a total bacteria count (MPN/100ml) ranging from 0 – 1100 MPN/100ml. Escherichia coli had the highest incidence of 80% followed by Staphylococcus aureus 66%, Salmonella species 48%, Pseudomonas aeruginosa 46%, Streptococcus sp 36%, Shigella species 36%, Klebsiella species 22%. The pH, conductivity, total dissolved solids, dissolved oxygen and sodium chloride levels ranged from 6.0 to 7.6, 8.4 to 188.4μS/cm, 4.2 to 94.2mg/l, 0.08 to 0.16mg/l and 0 to 0.4 respectively.
ConclusionThere is urgent need to intensify the monitoring of activities of this rapidly expanding industry and enforcing strict hygienic measures with a view to raising standards to improve packaged water quality.
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The mutational variety of the live-attenuated influenza vaccine proteome
More LessInfluenza viruses evolve rapidly, and for this reason the influenza vaccine needs to be updated every year. It is therefore important to identify where new mutations could be tolerated. To assess this, we asked which mutations could be identified in the proteins that had passed quality checks by being correctly folded, transported and assembled into influenza virus particles. We re-analysed mass spectrometry proteomics data obtained from the virus particles of genetically well-defined vaccine strains and identified point mutations within viral proteins. Point mutations were tolerated in virus particles at appreciable frequencies in proteins of both influenza A and B viruses, including HA, NA, M1, NP, NS1, PA and PB2. Structural analyses were used to assess the likely impact of this protein diversity on the molecular biology of the virus. As would be expected, mutations that were tolerated the virus particle generally occurred at sites that would not be expected to perturb protein function. We suggest that using proteomics to identify sites in viral proteins that either can or cannot tolerate mutations could inform influenza vaccine development by highlighting areas that have the potential for antigenic drift.
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Exploring foot-and-mouth disease virus antibody interactions using biolayer interferometry
Foot-and-mouth disease virus (FMDV) vaccines protect animals from infection by inducing antibodies. The level of neutralising antibody induced in response to vaccination (or infection), as measured by a virus neutralisation test, is an important parameter with regards to the level of protection afforded against subsequent challenge. However, in addition to overall titre, antibody avidity also represents a crucial metric when assessing the protective efficacy of antibodies. In this project we investigated the use of biolayer interferometry (BLI) to measure the avidity of FMDV antibodies to FMDV antigens. Antibodies targeting site I of the FMDV particle were detected using a commercially synthesised biotinylated peptide. In contrast, the entire antigenic landscape of the FMDV particle was represented by biotinylated FMDV capsids. The antigens were loaded onto Octet streptavidin biosensors at an optimal concentration prior to dipping into antibodies. The sera from different animals varied in avidity, reflecting the quantitative differences in avidity that exist between individual animals in response to FMDV vaccines. Interestingly, the Kdis values obtained for site I vs the entire capsid were different, supporting the importance of other sites beyond site I. Similarly, monoclonal antibodies targeting distinct, known antigenic sites on the capsid surface also resulted in different avidities. The BLI methodology reported here offers a useful approach by which to investigate the strength of antibody interactions at specific sites. In conjunction with recombinant technology, BLI will help aid in investigations into the relative importance of the different antigenic sites with regards to inducing a protective response.
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Genomic diversity of Bacillus anthracis in endemic settings: novel approaches and data
Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains endemic in many developing countries where it has significant impacts on the health and livelihoods of livestock-keeping communities. While the global genomic diversity of B. anthracisis is well characterised, few studies have quantified its diversity in endemic settings at more local scales, where this information could be critical for elucidating transmission dynamics and guiding control efforts. We collected samples from 526 anthrax-suspected animal carcasses between 2016 and 2018 in the Ngorongoro Conservation Area in northern Tanzania. Seventy five percent were confirmed positive through qPCR, suggesting that anthrax is responsible for a large proportion of sudden deaths in livestock in this area. A subset of positive samples were cultured for whole genome sequencing (n = 73), including multiple isolates from individual carcasses. All sequenced isolates belonged to the Ancient A lineage of B. anthracis, a common strain in southeastern Africa. No clear spatial clustering was observed, possibly reflecting extensive animal movement related to local nomadic practices. Moreover, high levels of within-host diversity were observed which suggests that cases commonly result from simultaneous infection with multiple strains. Additionally, we trialed a targeted sequence capture approach on 93 samples. This was successful in recovering >80% of the chromosomal genome at > 15X coverage from 60% of samples tested, thus representing a valuable tool for culture-free sequencing of this high-risk bacterium. Our work paves the way for integrating genomic data for B. anthracis into epidemiological studies and monitoring of control programs in endemic areas worldwide.
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Piperacillin/tazobactam resistance in a clinical isolate of Escherichia colidue to IS26-mediated amplification of blaTEM-1B
We identified a clinical isolate of Escherichia coli displaying an unusual, emerging phenotype; piperacillin/tazobactam (TZP)-resistant, 3rd generation cephalosporin-susceptible. Prior to treatment with TZP, a TZP-susceptible E. coli isolate was isolated from the same patient. Hyperproduction of a class A β-lactamase has previously been linked to this phenotype, but the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood.
Clonality of the two isolates was initially assessed with RFLP, and β-lactamase activity was determined using a nitrocefin assay. Both isolates were sequenced on Illumina and Oxford Nanopore Technology platforms and fitness assessed competitively. A plasmid construct containing the insertion sequence IS26was used to capture a translocatable unit (TU) in vitro.
The two E. coli clinical isolates were confirmed to be clonal, with the TZP-resistant isolate hyperproducing blaTEM-1. However, no promoter region mutations were identified in the TZP-resistant isolate. Hybrid assembly revealed that an ~11kb segment of DNA was excised from a IS26 flanked pseudo-compound transposon in the TZP-resistant isolate, forming a circular TU containing blaTEM-1. Multiple re-insertion events of the TU, mediated by IS26, led to tandem repeats of the TU within the chromosome, increasing the copy number of blaTEM-1. Excision and insertion events were confirmed via capture of the TU. Amplification of the TU in the TZP-resistant isolate incurred no significant change in fitness in different environmental conditions.
This study improves the understanding of the TZP-resistant, 3rd generation cephalosporin-susceptible phenotype in E. coli and antimicrobial resistance prediction of this phenotype from genotypic data.
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