- Volume 4, Issue 5, 2022
Volume 4, Issue 5, 2022
- Abstracts from Annual Conference 2021
-
- Poster Presentations
-
-
Diversity Analysis and Genome Mining of Microbial 16S rRNA Gene and Metagenomic Data for the Discovery of Novel Antibiotics
More LessThere is an urgent need for new antimicrobials due to constantly advancing antimicrobial resistance. Here, we worked with environmental samples from diverse habitats including different savannah and forest soils, volcanic caves, and termite mounds and assessed their microbial communities for the potential of biosynthesis of secondary metabolites. We analysed and compared microbial composition by applying the QIIME2 pipeline to 16S rRNA gene data. We focused on the abundance of Actinobacteria and Streptomyces as they are important producers of antimicrobials. Out of the samples analysed, the highest abundance of Actinobacteria was found in termite mound and volcanic cave samples. Moreover, the termite mound samples also had the highest abundance of Streptomyces. When comparing microbial composition, soil samples and termite mound samples each formed their own clusters, but volcanic cave samples appeared more dispersed. We assessed the antimicrobial potential of a subset of samples by analysing metagenomic data to predict biosynthetic gene clusters (BGCs) using antiSMASH5.2.0, which resulted in over 800 hits per sample. This number was narrowed down by evaluating identified BGCs based on antimicrobial potential, completeness, size, presence/absence of regulatory and transport-related genes, and dissimilarity with known BGCs. This resulted in an average of 20 BGCs per sample. These BGCs will be subjected to further sequence-based analyses before attempting heterologous expression. Following successful expression, antimicrobial potential will be assessed by screening for growth inhibition of multidrug resistant E.coli strains and the ESKAPE pathogens.
-
-
-
Investigation of the impact of food preservatives on avian pathogenicEscherichia coli(APEC) and their role in driving zoonotic disease
More LessThe excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial antimicrobial resistance. Globally, antibiotics are also widely used as growth supplements in livestock. This has led to concerns regarding use of human-use antibiotics in food and food-producing animals. In more recent times organic acids such as propionic acid (PA) and formic acid (FA) have been used as alternative antimicrobials or preservatives in place of antibiotics.
PA is a short chain fatty acid naturally abundant in the human and animal intestine as a breakdown product of non-digestible carbohydrates. In the human intestine it plays important roles in regulating the immune response in the human body. Recently, a study has shown that exposure of a Crohn’s Disease associated bacterial pathotype, adherent-invasive Escherichia coli(AIEC),to PA significantly altered its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm-formation.
AIEC are both evolutionarily and phylogenetically related to avian pathogenicEscherichia coli (APEC). PA and FA use is widespread in chickens a known source of zoonotic disease. Our results indicate that virulence of some APEC strains is increased by exposure to alternative antimicrobials such as PA and FA. This included increased adhesion of APEC strain E. coli 601 to human intestinal epithelial cells after exposure to FA which could be a potential risk of zoonotic disease. Further investigation of APEC strains is currently underway in a fermentation model of the poultry gut. This approach will improve our understanding of how commonly used.
-
-
-
Diversity and dynamics of particle-associated and free-living bacteria in eelgrass (Zostera marina) bed along the coast of Japan
More LessAs the seagrass leaves remain underwater, the primary source of leaf microbes is considered to be seawater heterotrophic bacterioplankton, which possess the ability to degrade biopolymers and are known to attach to surface and form biofilms. In this study, 16S rRNA gene amplicon sequencing was used to assess bacterial diversity and dynamics of the particle associated (PA) and free-living (FL) fraction of the seagrass-covering seawater (inside) and bulk seawater (outside) among different seagrass bed around Japan. Samples were collected from the three Zostera marinabeds (Ikuno-shima Is., Hiroshima; Nanao Bay, Ishikawa; Mutsu Bay, Aomori Prefecture) around Japan during summer (June-August 2015; July 2016). Prokaryotic DNA was extracted from samples using a FastDNA spin kit according to the manufacturer’s protocol. After extracting DNA, 16S ribosomal RNA (16S rRNA) genes were sequenced by Illumina Miseq platform. The Results showed that PA bacterial communities had a higher (p <0.001) diversity than FL ones. Compared to the outside of the seagrass bed, the inside had lower diversity both in PA and FL fraction. Taxonomic analysis revealed a different community composition between lifestyle (PA vs FL) and sampling point (inside vs outside). Differential abundance analysis showed that PA were significantly enriched in a diversity of Cyanobacteria (Synechococcaceae), Saprospiraceae and Hyphomonadaceae. Conversely, FL were more abundant in Gammaproteobacteria (including Halomonadaceae, Alteromonadaceae), Microbacteriaceae, Campylobacteraceae, Pelagibacteraceae,Acidimicrobiia(OCS155). The present data provide a comprehensive description of the PA and FL microbial community in the seagrass bed and can be useful for better understanding the seagrass microbe interactions.
-
-
-
Teaching Microbial Genomics in the COVID-19 age: principles, theory, and practice
More LessThe importance of genomics in the COVID-19 age cannot be overstated and genomics has a key place in the advanced undergraduate microbiology curriculum. The COVID-19 pandemic, and its attendant lockdowns, have necessitated a change in the delivery of our microbial genomics module. The key challenges for delivering a remote computer-based genomics module, are ensuring student engagement and learning; and supporting the technical aspects of remote computer work.
In the absence of in-person classes, we have adapted our material covering the principles and theory of microbial genomics, to asynchronous videos and synchronous online workshops. Practical training for all undergraduate microbiologists in the UK has been severely reduced, which has forced us to focus skills training towards computational aspects of genome assembly, analysis, and interpretation. The set up and implementation of a robust and scalable Linux-based genome analysis pipeline for students presents many challenges: from organising remote access to computer clusters; software support; and managing hardware.
Assessment for the module is based on the demonstrating learning and skills development through the analysis of SARS-CoV2 genomes to identify spike protein variation and mapping this to published structures. Terminal assessment is through the analysis of newly sequenced bacterial genomes and the preparation of a genome report suitable for publication.
We will outline the implementation and management of our Microbial Genomics module as a model for computer-based skills training for undergraduate microbiologists. Furthermore, we will discuss the impact of the COVID-19 pandemic on the module and the opportunities it has presented.
-
-
-
Improving phage genome annotation to understand phage biology: the case of Pseudomonas aeruginosa LES prophages
More LessPseudomonas aeruginosais an important opportunistic pathogen, causing nosocomial infections. The Liverpool Epidemic Strain (LES), a major cause of mortality and morbidity in cystic fibrosis patients, harbours five prophages associated with increased fitness and survival in models of infection. However, ~76.5% of the LES prophage genes are hypothetical proteins. Also, little is known about the LES prophage interactions with the lysogen and other prophages. In this study, we used the VIral Genome Annotation (VIGA) pipeline to re-annotate the LES prophage genomes and improve the prediction of gene function. The RefSeq viral proteins database was used for homology-based gene prediction and the Prokaryotic Virus Orthologous Genes (PVOGs) and Reference Viral DataBase (RVDB) were used for HMM-based protein function prediction. InterProScan 5.47-5.82 and Infernal 1.1.3 (with Rfam 14.3) were applied to enrich the gene function prediction for all genes and ncRNA elements, respectively. The number of putative coding sequences had increased by 1.17-1.43 times. Multiple genes related to DNA recombination and host cell lysis were identified in this reannotation. Also, we have identified new ncRNA elements in these prophages, such astRNAs in prophages 2 and 5 and a putative regulatory ncRNA, a Hammerhead-II ribozyme, in prophage 4. All this new information will be combined with data from future RNAseq experiments to map the expression profiles of each LES prophage under inducing and non-inducing conditions to characterise interactions between the prophages and their lysogen host.
-
-
-
Development of CRISPR/Cas9-based Novel Vaccines against Poultry Viruses
More LessVaccines remain the primary means of disease prevention throughimmunisationschemes in the poultry sector. Novel approaches in vaccine development, such as reverse genetic systems and genome editing technologies (i.e. CRISPR/Cas9), are currently being utilized to overcome challenges in establishing an immunogenic platform that is safe and capable of inducing long-term immunity. The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats associated protein nuclease 9) technology offers an effective, fast and simple novel approach to edit genomes for the development of viral vectors against poultry diseases such as Newcastle disease (ND), avian influenza (AI) and infectious bronchitis (IB). In this study, we demonstrate the application of CRISPR/Cas9-based genome editing to generate recombinant viral vectors that express the F gene of NDV. Validation of gene integration, protein expression, and insert stability was carried out by PCR, immunostaining and Western blot analysis, respectively. This approach offers an efficient platform for the generation of multivalent recombinant vaccines that can provide simultaneous protection against major poultry diseases.
-
-
-
How do bacteria change their coats? Structural analysis of acyltransferases involved in O-antigen modification
More LessAcyltransferase-3 (AT3) domain-containing proteins are involved in acylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, antimicrobial resistance, and antibiotic biosynthesis. Despite this, the mechanism of action is largely unknown. AT3 proteins fromSalmonellaspp. are responsible for acetylation of lipopolysaccharides which can generate a specific immune response upon infection. Here we analysed two AT3 proteins fromSalmonellaspp., some differences exist but both contain an integral membrane AT3 domain fused to a periplasmic SGNH domain. Identification of essential residues from each domain suggests both domains are required for acylation.
The crystal structure of the SGNH domain and periplasmic linking region was determined. Novel structural features are seen in comparison to other SGNH domains. In particular, the periplasmic linking region is structured and forms an extension of the SGNH domain (SGNH-extension). Removal of the SGNH-extension suggests that this region is important for stability of the SGNH domain. The structure of the SGNH-extension suggests the SGNH domain is in close proximity to the acyltransferase domain and the domains may interact. In silicoco-evolution analysis, used to make predictions about the structure of both domains, suggests likely inter-domain interactions. This analysis also predicted which transmembrane helices in the acyltransferase domain interact giving an insight into the overall structure.
Combining these data we propose a refined model of AT3-SGNH proteins which enhances our understanding of the mechanism and function of AT3 proteins required for modification of cell-surface carbohydrates.
-
-
-
genomeRxiv: a microbial whole-genome database and diagnostic marker design resource for classification, identification, and data sharing
genomeRxiv is a newly-funded US-UK collaboration to provide a public, web-accessible database of public genome sequences, accurately catalogued and classified by whole-genome similarity independent of their taxonomic affiliation. Our goal is to supply the basic and applied research community with rapid, precise and accurate identification of unknown isolates based on genome sequence alone, and with molecular tools for environmental analysis.
The DNA sequencing revolution enabled the use of cultured and uncultured microorganism genomes for fast and precise identification. However, precise identification is impossible without
1. reference databases that precisely circumscribe classes of microorganisms, and label these with their uniquely-shared characteristics
2. fast algorithms that can handle the volumes of genome data
Our approach integrates the highly-resolved classification framework of Life Identification Numbers (LINs) with the speed and computational efficiency of sourmash and k-mer hashing algorithms, and the precision and filtering of average nucleotide identity (ANI). We aim to construct a single genome-based indexing scheme that extends from phylum to strain, enabling the unique and consistent placement of any sequenced prokaryote genome.
genomeRxiv includes protocols for confidentiality, allowing groups to identify and announce the identities of newly-sequenced organisms without sharing genome data directly. This protects communities working with commercially- and ethically-sensitive organisms (e.g. production engineering strains, potential bioweapons, and to enable benefit sharing with indigenous communities).
genomeRxiv will also provide online capability to design molecular diagnostic tools for metabarcoding and qPCR, to enable tracking of specific groupings of bacteria directly in the environment.
-
-
-
Regulatory cascade in SaPI activation
Staphylococcal pathogenicity islands (SaPIs) are mobile genetic elements encoding superantigens and other toxinsand are induced for excision, replication, packaging and intercell transfer by phage-encoded anti-repressors that counter the SaPI master repressor. Though SaPI induction has heretofore been assumed to be the exclusive province of helper phages, we report here the remarkable discovery that one of the SaPIs, SaPI3, can instead be induced only by a second, co-resident SaPI, which must first be induced by a phage. This induction cascade thus represents intricate regulatory triad; SaPI3, the beneficiary of this intracellular largess, is the prototype of a hitherto uniquely immobile SaPI lineage. We report that members of this lineage are controlled by a novel regulatory module and are induced by a highly conserved but previously uncharacterised SaPI protein. SaPI3 and its cousins are thus SaPI satellites, just as most other SaPIs are phage satellites.
-
-
-
Amoeba & Biofilms in UK Chlorinated Drinking Water Distributions Systems: Impact on Water Safety
More LessAmoeba-related diseases have been related with the presence of certain amoebas in domestic water, including drinking water. Biofilms in drinking water distribution systems are able to support amoeba growth by providing a food source and protecting them against disinfectants. Additionally, amoeba growth can be favoured by warm temperatures and climate change appears to contribute to its geographic spread.
The presence of amoeba and its association with potential pathogenic bacteria was studied in a real-scale chlorinated DWDS. The test facility comprised three independent pipe loops fed with water from the local water supply and for this study a varied flow hydraulic profile was applied based on daily patterns observed in real UK distribution networks. The daily regime was repeated for a biofilm growth phase of 30 days. Amoeba viability was tested by a culture-based method, non-nutrient agar (NNA)-E. coli plates, and then confirm by qPCR using specific primers to detect species of amoeba including Naegleria and Acanthamoeba. Amplicon sequencing of the 16s rRNA gene was used to characterise the biofilm and planktonic bacterial communities.
Several amoeba species belonging to the genera Acanthamoeba, Vermamoeba and Naegleria were identified in 30-day old biofilm samples. While the bacterial communities in biofilms were dominated by Variovorax, Pseudomonas and Aquabacterium. This study yielded new insights on the dynamics of amoeba and bacterial communities in DWDS. However, more research is required to accurately establish the impact of these inter-kingdom associations on human health.
-
-
-
Antimicrobial resistance: revisiting the mechanisms of resistance
Resistance to antibiotics persists as a critical challenge in public health. Currently, the emergence of multi-drug resistant (MDR) bacteria is a primary concern globally, resulting in a dramatic increase in epidemiological relevance and importance of nosocomial and chronic infections. Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae has recently been classified as critical in the World Health Organization (WHO) priority pathogens. Among these bacterial pathogens, resistance seems to be a natural trait. The acquisition and development of resistance by bacteria is through several mechanisms. The genetic background and intrinsic resistance mechanisms largely contribute to competitive advantage and resistance in a highly resistant pool. The acquisition of resistance genes driven by mobile genetic elements (MGE) and several biochemical mechanisms also plays a central role in resistance development among pathogenic bacteria. This review discussed the recent underlying multiple resistance mechanisms among the priority pathogens. This review also provides an up-to-date regional epidemiological data and implications of antimicrobial resistance. Given the severity of infections caused by these bacteria, their less susceptibility to the available antimicrobials, and the limited antimicrobial arsenal to treat these pathogens, current insight on resistance mechanisms becomes timely and highly relevant. This information will help develop better therapeutic strategies against resistance microbes, especially those of urgent priority.
-
-
-
Beyond fluorescein: Use of fluorescent protein calibrants for direct and absolute quantification of protein production in synthetic biology
More LessWhile inter-lab calibration standards are approaching mainstream usage in synthetic biology, such calibrations are not in fact sufficient for absolute protein quantification required for modelling synthetic circuits. Fluorescein-based calibration of plate reader and flow cytometry instruments allows the measurement of green fluorescent protein (GFP) in synthetic cells to graduate from arbitrary units to calibrated units, but retains important caveats. Fluorescein is only a good calibrant for green FPs, leaving other FPs uncalibrated, and only provides conversions to units of brightness, not to molecule numbers.
Ideal assay calibrants in molecular biology consist of the same molecule as the one to be measured – in this case, a purified preparation of the appropriate fluorescent protein. Here we show that by using purified FP calibrants, all protein species in a synthetic circuit can be quantified in absolute terms using no advanced instrumentation. We develop a SEVA (Standardised European Vector Architecture)-based expression vector that allows the high-level production of soluble protein and describe a straightforward 2-day protocol for the purification of micrograms of FP, followed by a calibration that relates fluorescence activity to protein mass.
We validate this protocol by calculating conversion factors for a panel of commonly-used FPs including superfolder GFP, mCherry, mScarlet-I and mTagBFP2 on multiple laboratory instruments, and use these calibrations to debug synthetic circuits. We also demonstrate that the suspected bias of the presence of mCherry on OD600 measurements is real, but in practice requires extreme overexpression (~100,000 proteins per cell) to have a meaningful impact on cell density estimates.
-
-
-
Investigation of the Localisation of FtsZ in Pathogenic E. coli Upon Bacteriophage Addition in a Human Cell Model as a Biomarker for Antibacterial Agents
More LessAntimicrobial resistance is a growing problem worldwide and has created a need for novel antibacterial agents and strategies. Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Agents that block cell division have been shown to mis-localise FtsZ, including the bacteriophage λ encoded Kil peptide, resulting in defective cell division and a filamentous phenotype, and therefore FtsZ may be an attractive target for new antimicrobials.
In this project, we are interested in studying the localisation of FtsZ in pathogenic E. coli in the presence and absence of human cell cultures, in order to establish how and if this localisation changes upon infection. We are also interested in whether bacteriophages specifically attacking pathogenic E. coli have an effect on the localisation of FtsZ in a human cell environment and want to study the mechanism of this process. We have observed E. coliFtsZ localising to the cell midbody as a ring in both a K12 strain and in the K1 strain EV36 using confocal microscopy. These strains were used to infect human cerebral microvascular endothelial cells (hCMEC/D3) to create a meningitis model. We will present our results showing the effect of the Kil peptide and bacteriophages on this localisation within the model system, in an effort to validate FtsZ as a potential biomarker for antibacterial agents.
-
-
-
The ecology and antimicrobial resistance of Staphylococci colonising neonates
Coagulase Negative Staphylococci (CoNS) are common commensals of human skin, account for nearly 20% of the microbiota in infants and are thought to promote early immune responses in healthy babies. However, CoNS are opportunistic pathogens and in the UK between 2005 and 2014 were responsible for 57% of episodes of Late Onset Sepsis (LOS). In neonatal intensive care units (NICUs) this is a major concern and antiseptics are used to prevent vascular catheter infections. Chlorhexidine (CHX) and Octenidine (OCT) are the most common agents used for skin antisepsis, but evidence is emerging of antiseptic tolerance amongst CoNS.
We undertook a longitudinal survey of CoNS from skin and rectal swabs isolated from babies in two NICUs from countries with different antiseptic regimens (UK and Germany). Over 1000 isolates were characterised for antimicrobial susceptibility and sequenced. The most frequent species isolated were S. epidermidis and S. haemolyticus with similar strain types present in both units. Reduced susceptibility to CHX and OCT was observed in UK isolates (where CHX is used), compared to German isolates (where OCT is used).
Analysis of genome data using GWAS and clustering techniques has identified loci associated with antimicrobial susceptibility. Comparison of isolates taken on admission and thereafter, demonstrated that babies acquired isolates with decreased antiseptic tolerance after admission. This data provides new information about the phylogeny of CoNS in NICUs and suggest different potentials for selection of resistance between antiseptics commonly used in neonatal care.
-
-
-
Vegetables and rhizospheres as the reservoir for extensively drug-resistant (XDR) Pseudomonas aeruginosa
More LessPseudomonas aeruginosais an urgent threat pathogen due to its evolving resistance to multiple antibiotics. Agricultural soil and plants are the vast reservoirs of this much-dreaded opportunistic bacterium. A few human isolates of P. aeruginosa are known to infect plants and insects. However, there is no report on the occurrence of multi-drug resistant P. aeruginosa in edible vegetable crops. This study compared 18 P. aeruginosa isolates from the rhizosphere and endophytic niches of four different vegetable crops (cucumber, tomato, eggplant, and chili) with three known clinical strains. All the isolates were tested for virulence traits such as resistance to various antibiotics classes, motility, biofilm, and production of virulence factors (rhamnolipid, pyocyanin, hemolysin, proteases, and lipases). Hierarchical clustering based on Ward minimum variance with Manhattan distance matrix grouped the strains into three clusters based on their phenotypic traits. Strains were exhibiting the highest virulence co-clustered with the human pathogenic isolates. These strains were resistant to cephalosporins, aminoglycosides, macrolides, nitrofurans, tetracyclines, and sulfonamides. These extensively drug-resistant (XDR) strains were only susceptible to polymyxin (colistin) and quinolone (cephalosporin). This study shows that the virulence traits are shared between plant- and human-isolates of P. aeruginosa. More importantly, the occurrence of XDR strains in vegetable crops is a serious global threat.
-
-
-
Exploring bacterial diversity via a curated and searchable snapshot of archived DNA
The open sharing of genomic data provides an incredibly rich resource for the study of bacterial evolution and function, and even anthropogenic perturbations such as the widespread use of antimicrobials. Whilst these archives are rich in data, considerable processing is required before a biological question can be addressed. Here, we have assembled, quality controlled and characterised 661,405 bacterial genomes that were in the European Nucleotide Archive (ENA) at the end of November of 2018, using a uniform standardised approach. A searchable index has been produced, facilitating the easy interrogation of the entire dataset for a specific gene or mutation. Our analysis shows how uneven the species composition is within this database, with just 20 of the total 2,336 species making up 90% of the high-quality genomes. The over-represented species tend to be acute/common human pathogens, often aligning with research priorities at different levels from individuals with targeted but focussed research questions, areas of focus for the funding bodies or national public health agencies, to those identified globally as priority pathogens by the WHO for their resistance to front- and last-line antimicrobials. Whilst this is a rich resource which often forms the context or references for multi-‘omic’ studies and supports discovery research in many domains, understanding the actual and potential biases in bacterial diversity depicted in this snapshot, and hence within the data being submitted to the public sequencing archives, is essential if we are to target and fill gaps in our understanding of the bacterial kingdom.
-
-
-
A study on the antibacterial efficacy of Halohydantoin-containing foams
More LessHeterocyclic N-halamine compounds such as 1-monochloro-5,5-dimethylhydantoin (MCDMH) and 1,3-dichlor-5,5-dimethylhydantoin (DCDMH) show antimicrobial activity and odour control. Both MCDMH and DCDMH stabilise and display oxidative chlorine (Cl+1) and, in aqueous environments, generate hypochlorous acid (HOCl). It is well known that HOCl, produced by neutrophils as part of the respiratory immune response, is used as a defence mechanism against foreign bodies. This study aimed to investigate the antibacterial efficacy of proprietary foaming formulations containing mixtures of MCDMH and DCDMH provided by MedeSol Inc., as a potential delivery mechanism for HOCl. The efficacy of the foams containing active chlorine concentrations of 350 ppm and 1050 ppm was tested against single and multi-species bacterial mixtures of S. aureus, P. aeruginosa, E. coli, E. faecalisand K. pneumoniae. Cellulose filters were contaminated with single/multispecies bacterial suspensions and left to air-dry; the two foam concentrations were applied for 15, 30 and 60 seconds at a time. Samples were taken and the total viable count method was employed to determine antibacterial efficacy of the foams. Following the use of both foam concentrations, no colonies were recovered at any time-points in both single and multi-species experiments. In contrast, the negative controls where the foam application was omitted, resulted in high numbers of bacterial colonies. MCDMH-containing foams have very strong antibacterial potential as a result of active chlorine release. Further studies are needed to determine concentration dependence and use in other applications including hand sanitation.
-
-
-
Investigation of the influence of plant tissue damage on the attachment of Listeria to fresh salad produce
More LessVegetables and fruits are an important for a healthy life due to them containing nutritionally important minerals and vitamins. Green salads (spinach, lettuce) are popular convenience consumer products, however evidence is increasing that these ready to eat salads have been identified as a source of foodborne illness. There are many pathogenic bacteria which are transmitted by food that can cause serious disease. One of the most important is Listeria monocytogenesbecause it can cause invasive infections and especially because of its ability to grow at low (food refrigeration) temperatures. The aim of this study is to investigate whether compounds released from damaged fresh salad leave have an effect on Listeria growth and virulence. The results in this report show that Listeria was highly responsive to the salad extracts and that growth and biofilm formation were both increased compared to un-supplemented control cultures. Plant derived chemicals also stimulated Listeria growth in serum-SAPI medium. The results also showed that Listeria treated with salad extract was more virulent in a Galleria mellonella infection model.
-
-
-
How to sequence 10,000 bacterial genomes and retain your sanity: an accessible, efficient and global approach
Blanca Perez-Sepulveda, Darren Heavens, Caisey Pulford, Alexander Predeus, Ross Low, Hermione Webster, Gregory Dykes, Christian Schudoma, Will Rowe, James Lipscombe, Chris Watkins, Benjamin Kumwenda, Neil Shearer, Karl Costigan, Kate Baker, Nicholas Feasey, Jay Hinton, Neil Hall and The 10KSG ConsortiumNon-typhoidal Salmonella(NTS)are typically associated with enterocolitis and linked to the industrialisation of food production. In recent years, NTS has been associated with invasive disease (iNTS disease) causing an estimated 77,000 deaths each year worldwide; 80% of mortality occurs in sub-Saharan Africa. New clades of S. Typhimurium and S. Enteritidis have been identified, which are characterised by genomic degradation, altered prophage repertoires and novel multidrug resistant plasmids. To understand how these clades are contributing to the burden and severity of iNTS disease, it is crucial to expand genome-based surveillance to cover more countries, and incorporate historical isolates to generate an evolutionary timeline of the development of iNTS.
We developedand validateda robust and inexpensive method for large-scale collection and sequencing of bacterial genomes. The “10,000 Salmonella genomes” project established a worldwide research collaboration to generate information relevant to the epidemiology, drug resistance and virulence factors of Salmonellae using a whole-genome sequencing approach. By streamlining collection of isolates and developing an efficient logistics pipeline, we gathered 10,419 clinical and environmental isolates from collections in low and middle-income countries within six months. Genome sequences are now available for isolates from 51 countries/territories dating from 1949 to 2017, with ~80 % representing African and Latin-American datasets. Our method can be applied to other large sample collections that require maximisation of resources within a limited timeframe. Detailed genome analyses are in progress and it is hoped that the resulting data will contribute to public health control strategies in low and middle-income countries.
-
-
-
Clostridioides difficile surveillance at the University Hospital in Košice reveals high prevalence of the ribotype 176
BackgroundClostridioides difficile infection (CDI) has become a serious health problem worldwide in recent years, the severity of which lies in the ability to spread epidemically in hospitals and in frequent diseases relapses. CDI Surveillance was performed at the Louis Pasteur University Hospital in Košice from January to February 2020 to analyse the molecular characteristics ofC. difficile(CD) isolates from local patients with CDI.
MethodsCDI was initially diagnosed using the C. difficile rapid test (for enzyme GDH and toxin A/B). A total of 36 stool samples (29 GDH and toxin positive, 7 GDH positive and toxin negative) were cultured anaerobically on selective media (Brazier’s agar). Culture was positive for CD in 31 samples. Bacterial DNA was extracted from all CD isolates. Genes tcdA, tcdB, cdtA and cdtB encoding toxin A, toxin B and binary toxin were detected by multiplex PCR and ribotypes of CD were analysed by capillary electrophoresis-based PCR.
ResultsMolecular typing showed that toxin A as well as toxin B was confirmed in 30 of 31 isolates, binary toxin in 18 isolates. Ribotype 176, characterized by production of all 3 toxins, was the most prevalent and was detected in 18 isolates (58%). Toxin A and toxin B producing ribotypes 001, 014 and 020 were also confirmed.
ConclusionThe high incidence of epidemic ribotype 176 with higher capacity to spread in a hospital setting emphasises the need to implement strict epidemic measures and the importance of implementing continuous surveillance programmes for CDI in Slovakia.
-