bluuebunny/crossref_metadata_2025 huggingface dataset:
Shiga toxin-producing E. coli (STEC) isolated from wild mammals in Portugal
Authors: Dias Diana , Costa Sávio , Baraúna Rafael , Fonseca Carlos , Caetano Tânia , Mendo Sónia
Published: May 2022

Background

Zoonoses 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.

Methods

Faecal 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.

Results

Cultivable 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.

Conclusions

Our results show that wild animals are reservoirs of STEC, potentially pathogenic to humans.
Intraspecies-Resolution Metabarcoding: automated primer design and a plant pathology case study
Authors: Pritchard Leighton , Humphris Sonia , Campbell Emma , Watts Lauren , Haynes Ed , Pufal Hollie , Cahill Greig , Davey Triona , Elphinstone John , Toth Ian
Published: May 2022

We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis.

Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible.

To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp (https://github.com/widdowquinn/find_differential_primers [https://github.com/widdowquinn/find_differential_primers]) that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism.

We aimed to survey geographical distribution and pathogen transfer of the widespread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.
Epigenetic Modification Impacts on Gene Regulation in Streptococcus pneumoniae
Authors: Althari Yasmeen , Chen Yongkui , Haigh Richard , Oggioni Marco
Published: May 2022

Methylation by the type I restriction modification system (RMS) SpnIII in Streptococcus pneumoniae is hypothesised to regulate gene expression via epigenetic changes (Manos). The phase variable SpnIII generates six host specificity determinants (hsdS) alleles through site-specific recombination (DeSteCroix), each allele correspond with different methylation pattern.

In addition to known functions of the RMS in restricting phage infection (Furi) and transformation (Kwun), we have now tested impact on gene expression. RNAseq was used to analyse S. pneumoniae strains expressing a single spnIII allele (spnIIIA, spnIIIB or spnIIIE) to determine differences in gene expression profiles. The data have identified six genes which show differential expression and have a methylation site mapping to their predicted promoter region. Three synthetic promoters with the wild type and altered methylation target site were cloned in front of a luciferase gene in strains expressing a single spnIII alleles. For the first three sets of constructs analysed, data indicate that the methylated promoters show a three- to twenty-fold higher activity compared to non-methylated promoters. Results obtained were further confirmed by qPCR analysis. Preliminary data using drugs targeting DNA topoisomerases indicate that the mechanism by which methylation impact gene expression could be related to DNA topology. These data demonstrate for the first-time RMS dependent variations in gene expression and propose an alternative mechanism for this epigenetic regulation.

References:

* Manso, Nature Communication, 2014; 5:5055.

* De Ste Croix, J. Bacteriol. 2019; 201(15). pii: e00233-19.

* Furi, J. Bacteriol. 2019; 201(19). pii: e00370-19.

* Kwun, Nucleic Acids Res. 2018; 46(21):11438-11453.
Environment and the Evolutionary Trajectory of Horizontal Gene Transfer
Authors: Bhatia Rama , Kirit Hande , Bollback Jonathan
Published: May 2022

Gene acquisition through horizontal gene transfer (HGT) may either exert a beneficial, neutral, or deleterious fitness effect on the recipient cell thereby determining the evolutionary fate of the newly transferred gene. The distribution of fitness effects (DFE) thus is a fundamental predictor of the outcome of an HGT event.

The environment plays a considerable role in altering the fitness of a horizontally transferred gene. We have studied the DFE of genes transferred from Salmonella enterica serovar Typhimurium to Escherichia coli in six environments, that potentially represent the conditions experienced by the two species. The data suggests high variability, with some genes becoming deleterious in one environment, while becoming neutral or even beneficial in another, suggesting that fluctuating environments may increase the likelihood of HGT.

The present study focuses on the DFE of genes transferred from Escherichia coli to macrophage-associated Salmonella Typhimurium strain 4/74 in four environments, that mimic the conditions inside a macrophage cell. In addition to the external environment, we are also looking at, how changes in the intrinsic environment of a cell, after an HGT event, could affect fitness. Functional similarity of the horizontally transferred gene to the endogenous copy can cause an imbalance due to increased protein dosage, thereby leading to a negative fitness effect. By comparing the growth rates of each ortholog gene with the ‘wild type’, in the four environments, we can elucidate when gene dosage acts as a barrier to HGT, helping us to understand the relationship of environmental quality and HGT, which is of evolutionary importance. We have identified 12 genes showing dosage dependent effects across the four environments, with some genes showing environment specificity, indicating an increased intolerance of the recipient to high protein dosage, making it a significant barrier to HGT.

A preprint is available on bioRxiv https://doi.org/10.1101/2022.04.01.486712
Automated pseudogene detection reveals insights into historical gene sharing dynamics in prokaryotes
Authors: Dimonaco Nicholas J , Aubrey Wayne , Clare Amanda , Kenobi Kim , Creevey Chris
Published: May 2022

In recent years it has become apparent that prokaryotic genomes contain large numbers of pseudogenised genes which may provide valuable insights into the recent functional history of an organism. However, pseudogenes are difficult to detectab initioand are not routinely reported by gene prediction tools.

We present StORF-R(Stop-ORF-Reporter), a tool that takes as input an annotated genome and returns putative missed genes (functional and/or pseudogenised) from the intergenic regions. We show that this methodology can recover gene-families that the state-of-the-art methods continue to misreport or completely omit.

We applied StORF-R to the intergenic regions of2,665E. coligenomes and found on average 244 previously missed pseudogenised genes (with in-frame stop codons) per genome, many of which had high scoring similarity to known Swiss-Prot proteins. Many of these pseudogenised genes form widespread gene families across E. coli strains.

To investigate if this phenomenon exists in other taxa we further applied the methodology to 44,048 bacterial genomes representing 8,244 species from Ensembl. This revealed manygene-families spanning multiple species with large (>10,000) numbers of copies of both intact and pseudogenised versions. Many of these families had only previously been reported in a single or few genomes, though we detected many hundred pseudogenised versions with StORF-R, changing our understanding of how widespread these genes truly are.

These pseudogenised genes represent a pangenomic ‘graveyard’ which may alter our understanding of the definition of core and accessory genes for many species.
Covariation analysis of the master bacterial DNA replication initiator DnaA
Authors: Dobby Laura , Murray Heath
Published: May 2022

Bacterial 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.
Sessile Clostridioides difficilecontribute towards recurrent C. difficileinfection
Authors: Normington Charmaine , Moura Ines , Byrant Jessica , Ewin Duncan , Ford Christopher , Wilcox Mark , Buckley Anthony
Published: May 2022

C. difficile, an anaerobic spore-forming intestinal pathogen, produces up to three toxins that cause host cell damage resulting in disease, C. difficileinfection (CDI). Therapies include antibiotic treatment; however, up to 30% of cases fail primary therapy, resulting in recurrent disease, which increases patient morbidity and places a burden on worldwide healthcare systems. We have little understanding of why these therapies fail. Using a clinically validated in vitro gut model, we assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. During induction of simulated CDI, C. difficile spores and vegetative cells became associated with the colonic biofilm microbiota. Vancomycin treatment did not effectively remove the biofilm C. difficile cells and recurrent infection was observed. Additionally, vancomycin therapy followed by faecal microbiota transplant did resolve the recurrent infection, but the biofilm C. difficile cells remained unaffected. In a biofilm transfer experiment, we showed that transferring biofilm encased C. difficile cells into a C. difficile naïve (but CDI susceptible model) induced CDI. Furthermore, we show that members of the biofilm community can impact C. difficile biofilm formation either acting in an antagonistic or synergistic manner. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.
Histopathological analysis of placental lesions caused by Chlamydia abortus 1B vaccine strain in vaccinated ewes
Authors: Caspe Sergio Gastón , Livingstone Morag , Wattegedera Sean , Aitchison Kevin , Underwood Clare , Milne Elspeth , Sargison Neil , Chianini Francesca , Palarea-Albaladejo Javier , Longbottom David
Published: May 2022

Background

Chlamydia abortus is one of the most diagnosed causes of infectious abortion in small ruminants. Infections can be controlled using the live, attenuated C. abortus strain 1B vaccine, which has been associated with infection and abortion in animals. This study aimed to compare the severity and the distribution of lesions caused by this vaccine strain (vt) with those resulting from a wild-type (wt) infection.

Methods

Two grossly affected and 1B-positive (by qPCR and RFLP analysis) placentas from a vaccinated sheep flock were analysed. Histopathological lesions and immunohistochemical labelling (IHC) were graded (increasing score from 0 to 5) according to their severity and distribution. Pathology in the vt infected placentas was compared with that in two wt infected placentas. Datasets generated for observed histological and pathological features were analysed using principal component analysis (PCA).

Results

Histopathologically, the lesions in both vt and wt-placentas presented as typical multifocal necrosuppurative placentitis, associated with vasculitis, mural necrosis, and thrombosis. IHC for C. abortus revealed intense staining with a multifocal distribution in most cotyledons in both vt and wt placentas. Comparison of the pathological lesions between vt and wt by PCA revealed a similar distribution and severity, revealing a strong association with features such as necrosis and inflammatory infiltration between vt and wt placentas. A weaker association with IHC was observed.

Conclusion

This study shows that the C. abortus attenuated 1B vaccine strain presents no significant differences in severity and distribution of pathological lesions from those typically observed following a wt infection.
Role of enteroaggregative E.coli induced activation of epidermal growth factor receptor in cultured human intestinal epithelial cell lines
Authors: Joon Archana , Chandel Shipra , Ghosh Sujata
Published: May 2022

Enteric pathogens exploit the versatility of cytoskeletal system for internalization into non-phagocytic cells, as a crucial step in their pathogenic life cycle. Enteropathogenic Escherichia coli(EPEC) andEnterohemorrhagic Escherichia coli (EHEC) were shown to form actin pedestals in host cells using type-III secretion system. Earlier, we reported that EAEC induced increase in intracellular calcium ions might have crucial role in F-actin rearrangements in INT-407 cells leading to its invasion. It was suggested that EGFR might contribute in Rck-mediated adherence and invasion of Salmonella in host cells. In the present study, we assessed the role of EAEC induced activated EGFR in human intestinal epithelial cell lines (INT-407 & HCT-15). The presence of activated EGFR was detected in membrane fractions ofeach cell line, infected with two different strains of EAEC (EAEC-T8 & EAEC-O42) separatelyfor 3h in presence and absence of Tyrphostin AG1478 (EGFR-inhibitor), by western immunoblotting using p-EGFR (Y1068) antibody. Adherence and invasion of EAEC-T8 to each cell line were checked in presence of Tyrphostin AG1478. Further, the effect of Tyrphostin AG1478 on cytoskeletal F-actin rearrangement of EAEC-T8 infected cells was assessed under confocal microscope following staining with TRITC-phalloidin. EAEC-T8 induced maximum increase in EGFR autophosphorylation at Y1068. Adherence and invasion of EAEC-T8 as well as this organism induced cytoskeletal F-actin polymerization were found to be inhibited in presence of Tyrphostin AG1478. Our study revealed that EAEC induced activated EGFR might play a major role in host cell adherence and cytoskeletal rearrangements leading to invasion of the organism in these cells.
Defining the pknH Transcriptional Network in the Animal Tuberculosis bacillus, Mycobacterium bovis
Authors: Kerins Anne , Farrell Damien , Gordon Stephen V.
Published: May 2022

The Mycobacterium tuberculosiscomplex (MTBC) is a group of bacteria that show more than 99% genetic identity, yet they diverge in their host preference and the severity of associated disease. Mycobacterium bovis causes the disease in cattle and poses an economic challenge, with the cost of Irelands TB Eradication Scheme predicted to exceed €90 million in 2020.

The aim was to focus on the RD900 and pknH gene network in MTBC members. ThepknH gene encodes a transmembrane serine threonine protein kinase, that plays a role in the regulation of signalling pathways in the mycobacterial cell and has been linked to virulence. The RD900 or “region of difference 900” contains the tbD2 and pknH2 genes. The part of the genome that contains this region varies substantially across MTBC members.

The results of RNA-seq analysis showed that there are statistically significant levels of differential gene expression in wildtype and knock-in M. bovis strains.

The RD900 analysis across the MTBC showed that there is variation in this important genomic region. Analysis carried out by Mata et al. 2020, showed that this RD900 region had been independently lost in different MTBC lineages and strains. M. bovis has retained the RD900 region and this is believed to be linked to its increased virulence.

The majority of the M . africanum strains were found to possess the standard pknH, tbD2, pknH2, embR gene arrangement as is found in M. africanum GM041182, the strain that is typically used as a reference.

Based on work of Mata et al., 2020.
A study on the antibacterial efficacy of Halohydantoin-containing foams
Authors: Nedelea Andreea-Gabriela , Robins Lori , Williams Jeffrey , Maddocks Sarah
Published: May 2022

Heterocyclic 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.
Assessing the interspecies relationship betweenPseudomonas aeruginosa and Staphylococcus aureus in mixed biofilms grown in alginate beads and collagen scaffolds
Authors: Khalid Ammara , Beeton Michael , Maddocks Sarah
Published: May 2022

Polymicrobial biofilms in chronic infected wounds harbour different bacterial species that interact with each other, competing or co-operating to survive. The two most common pathogens co-isolated from chronic wound biofilms are Pseudomonas aeruginosa and Staphylococcus aureus. Evidence from in vitrobiofilms models have shown these two bacteria interactand data suggests that P. aeruginosa inhibits the growth of S. aureusin mixed biofilms. This study aimed to assess the growth of these two species in a complex polymicrobial biofilm in a 3D matrix comprised of either alginate (1.5% w/v) or a collagen scaffold.

Using a five-species biofilm (S. aureus, P. aeruginosa, Citrobacter freundii, Enterococcus faecalisand Escherichia coli), with all bacteria inoculated at time zero, it was consistently observed that P. aeruginosa was not recoverable over a 72h period, with sampling every 24h. However, P. aeruginosagrew well if it was added to a pre-formed four-species biofilm. Further, P. aeruginosa was seen to inhibit the growth of S. aureus after 24h subsequent co-culture in the pre-formed biofilm, which resulted in the emergence of small colony variants of S. aureus. Interestingly when P. aeruginosawas co-inoculated in a four species biofilm that did not contain S. aureus,its growth was not inhibited, suggesting a competitive interaction between these two bacteria during establishment of the early biofilm. These data were consistent in alginate beads and collagen scaffolds. In a chronic wound P. aeruginosais regarded as a late coloniser and the phenomena observed in this study might be reflective of this.
Functional responses of neutrophils to Fusobacterium nucleatum subspecies
Authors: Muchova Maria , Kuehne Sarah A. , Grant Melissa , Chapple Iain , Hirschfeld Josefine
Published: May 2022

Introduction

Fusobacterium nucleatum is an opportunistic oral pathogen with five subspecies: animalis, fusiforme, nucleatum, polymorphumandvincentii. These play an important role in biofilms associated with chronic gum disease (periodontitis). Neutrophils form the first line of defence against these and other oral pathogens, employing a multitude of anti-microbial strategies, which, alongside bacterial killing, also cause collateral host-tissue damage. The immunogenic properties of F. nucleatum subspecies are poorly understood. The purpose of this study was to investigate specific neutrophil responses to all F. nucleatum subspecies.

Methods

AllF. nucleatumsubspecieswere grown planktonically overnight and inactivated using 10% neutral buffered formalin. Peripheral blood neutrophils (PBN) and neutrophil-like cells (differentiated from HL-60 cells) were challenged with inactivated bacteria (MOI 100) and production of reactive oxygen species (ROS) was quantified by Luminol/Iso-luminol enhanced chemiluminescence assays. Release of neutrophil extracellular traps (NETs) was quantified fluorescently using Sytox-green. Additionally, phagocytosis of fluorescently labelled F. nucleatum subspecies was analysed in both cell groups by flow cytometry.

Results

In addition to differences in ROS generation by PBN and neutrophil-like cells in response to individual F. nucleatum subspecies, variations in the amount of extracellular DNA extruded during NETosis was evident. Moreover, distinct levels of phagocytic activity of both cell types will be shown.

Conclusion

F. nucleatum pathogenicity has been reported in oral as well as systemic diseases (colorectal cancer, cardiovascular disease). Understanding subspecies-specific effects of F. nucleatum on neutrophils will enhance our knowledge of the interactions between this bacterium and the host immune response and may help to identify new therapeutic targets.
Perfluorooctanoic acid (PFOA) sensitises E. coli to acid stress
Authors: Chugh Gaurav , O’Byrne Conor P , Abram Florence
Published: May 2022

Per-and Polyfluoroalkyl Substances (PFAS), which includes PFOS and PFOA, are recognized as the most important class of emerging contaminants due to their widespread presence in wildlife and humans, their environmental persistence, bioaccumulative potential, and toxicity. Low environmental pH conditions affect the state and physicochemical properties of PFAS, which in turn impacts on their toxicity. Sparse information is available regarding the effect of these compounds on microorganisms, and the possible associated knock-on effect on ecosystem services. The purpose of this study is to investigate the ability of E. coli to tolerate stress in the presence of PFAS compounds. To this end, we carried out phenotypic comparisons of E. coli exposed to a series of environmental conditions in the presence and absence of PFOA and PFOS. Quite remarkably, E. coli growth was not affected by the presence of PFOA and PFOS up to 500 mg/L. The survival of E. coli at pH3, however, decreased by more than three-fold when the medium was supplemented with PFOA. PFOA and PFOS were also found to decrease the growth rates of E. coli in minimal media in the presence of 0.75 and 1M NaCl. We are currently screening further stresses of environmental relevance with the aim to conduct molecular investigations to examine the mechanisms underpinning the effect of PFAS exposure on bacterial stress tolerance. This work will provide some insights into the impact of PFAS on microorganisms, which should shed new light on the assessment of the ecological effects of PFAS.
Sorghum beer production using Saccharomyces sp. isolated from palm wine as the pitching yeast and Garcinia kola as the bittering agent
Authors: Nwagu Tochukwu , Amadi OnyetugoChioma , Okon Bassey , Ozioko Anastesia , Nnamchi Chukwudi , Moneke Anene , Okolo Bartholomew , Agu Reginald , Ndubuisi Ifeany
Published: May 2022

Beer production in Nigeria is dependent on imported brewery yeasts and hops, which consequently increases the cost of beer production. The current study was conducted to determine the effect of using indigenous strains of Saccharomycessp. in place of brewer’s yeast, and Garcinia kola as the bittering agent on beer quality. Saccharomycesspecies strains were isolated from palm wine, andSaccharomycescerevisiae specie selected using nitrates and lysine assimilation test. IndigenousS. cerevisiaewas used for beer brewing at different pitching rates (1×107, 2×107, & 3×107) using brewer’s yeast as the control. The effect of various concentrations ofGarcinia kola(0.1 ml, 0.2 ml and 0.3 ml) on beer taste and quality was later determined; beer brewed with hops served as the control sample. The physicochemical analysis showed that the pH, specific gravity (SG) and free amino nitrogen (FAN) values of beer brewed with local S. cerevisiae strain were between 4.32 - 4.52, 0.962 - 1.008, and 81.80 - 263.64 g/L, respectively while the alcohol value was from 5.12 - 11.16 ABV. However, pitching with 1×107 cells gave the best product and was therefore used for further analysis. Beer samples from wort pitched with local yeasts and brewed withGarcinia kolagave good quality beer comparable to the standard product. pH values of the products were in the range of 3.18 - 4.41, SG (0.962 - 1.006), FAN (16.87 - 81.80g/l), bitterness (8.83 - 17.83 EBU) and alcohol (5.38 - 11.16 ABV).
Understanding the bug that make our drugs: Cellular responses to therapeutic protein secretion in E. coli
Authors: Seleiro Alice , Evans Caroline , Green Jeff , Lennon Chris , Stafford Graham
Published: May 2022

Production of therapeutic proteins is a $200 billion industry with E. coli being one of the main expression systems used and proteins often being secreted to the periplasm to produce disulphide bonds. However, little is known about how E. coliresponds to secretion of these therapeutic proteins into the periplasm under industrial conditions.

Proteomics and transcriptomics were conducted on cultures grown at high cell density in industrial production conditions to look at the effect of secreting a model therapeutic protein (scFv) to the periplasm compared to expressing the same protein in the cytoplasm.

82 proteins and 1653 transcripts were differentially expressed when overexpressing the scFv in different cellular compartments. ScFv secretion had a significant effect on expression of genes involved in the envelope stress response. However, while certain envelope stress pathways where upregulated upon secretion others were downregulated (including expression of some periplasmic chaperones). Scfv secretion also led to an increase in expression of genes involved in degradation of membrane proteins and the SEC secretion system, as well as iron transport, Outer membrane porins and flagella operons.

In future and current experiments, we are testing whether knocking out and overexpressing a selection of these genes improves secretion and folding of scFvs and other recombinant proteins.
Disruption of the mce operon from Streptomyces affects spore resistance and results in precocious germination
Authors: Addington Emily , Clark Laura , Hoskisson Paul
Published: May 2022

Streptomyces coelicoloris a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosisas an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Germination assays show that spores of Δmcegerminate earlier than WT S. coelicolor, and impression mounts indicate spore chains emerge earlier in the Δmcestrain. As spores of Δmce possess an altered spore coat, it was hypothesised they might show reduced resistance to stressors. Heat kill assays show that deletion of the mce operon results in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. Spores of the Δmcestrain are also less resistant to triclosan, and sulfobetaines. Preliminary results indicate that the mce operon of S. coelicolor may be a cholesterol importer, as Δmce shows reduced growth in the presence of cholesterol, compared to WT S. coelicolor.
Characterisation of the Rh50 protein from the ammonia-oxidising bacterium Nitrosomonas europaea
Authors: Bizior Adriana , Williamson Gordon , Mirandela Gaëtan Dias , Boeckstaens Mélanie , Hoskisson Paul , Marini Anna Maria , Zachariae Ulrich , Javelle Arnaud
Published: May 2022

The transport of ammonium through the cell membranes, an essential process in all kingdoms of life, is accomplished by the ubiquitous Amt/Mep/Rh superfamily of proteins. The functional context of Amt/Mep and Rh transporters is diverse: bacteria, fungi, and plants use Amt/Mep proteins to scavenge ammonium for biosynthetic assimilation, whereas mammals use the Rh proteins for ammonium detoxification in erythrocytes, kidney, and liver tissues. While RH50 genes are widespread in eukaryotes they are present in some prokaryotes: an example is a chemolithoautotroph Nitrosomonas europaeawhich gains all its energy from the oxidation of ammonia to nitrate.

While Amt/Mep/Rh proteins have divergent physiological functions, they are structurally very similar, which raises the important question about the universality of the transport mechanism. We have recently proposed an elegant new model for the mechanism of electrogenic ammonium transport in bacteria Amt protein: after deprotonation of NH4+ at the periplasmic side of the transporter, a previously undiscovered polar conduction route enables H+ transfer into the cytoplasm. A parallel pathway, lined by hydrophobic groups within the protein core, facilitates the simultaneous transfer of uncharged NH3. In this context, we propose to elucidate at the molecular level the mechanism of ammonium translocation through rhesus protein from Nitrosomonas europaeaand establish whether there is a universal mechanism for biological ammonium transport. Beyond the elucidation of a central biological process, this work has important medical implications, as some Rh mutations have been associated with human pathologies. We propose to demonstrate how specific Rh mutations affect the activity of the protein to establish the relationship between Rh malfunction and the associated diseases.
Uncovering the residues responsible for Dis3L2 specificity
Authors: Costa Susana M. , Matos Rute G. , Viegas Sandra C. , Arraiano Cecília M.
Published: May 2022

Exoribonucleases from the RNB-family of enzymes are widely distributed in nature. DIS3 is the eukaryotic homolog of the bacterial exoribonuclease II and is the only catalytic subunit of the core exosome complex. In humans, there are three members of DIS3 family that can be distinguished according to the sequence conservation of the active site:

DIS3, DIS3L (DIS3L1) and DIS3L2. Unlike its family counterparts, DIS3L2 does not interact with the exosome since it lacks the PIN domain, which is essential for the interaction with this multiprotein complex. Dis3L2 is involved in several cellular mechanisms, such as apoptosis, cellular differentiation and proliferation and its mutations have been associated with Wilms tumor formation and Perlman syndrome in children. Distinct studies on Dis3L2 enzyme unraveled a novel eukaryotic RNA decay pathway that challenged the models already established. Dis3L2 activity is stimulated by the addition of untemplated uridine residues to mRNAs, tRNAs, microRNAs, snRNAs among other classes of RNA.

The first insight on the uridylation involvement in controlling the stability of poly(A)-containing mRNAs was reported in S. pombe. However, the precise mechanism of action of this enzyme is not yet fully understood. In this work, the activity of fission yeast Dis3L2 mutant proteins was analyzed over different RNA substrates. The aim was to characterize the amino acid residues that distinguish Dis3L2 substrate specificities regarding its family homologues, namely the preference for uracil residues. The results show that some of the mutant Dis3L2 ribonucleases lose or acquire activity regarding the degradation of different RNAs. Furthermore, this will enable us to understand the mechanism of action of Dis3L2 and its function in different eukaryotic cells.
The Rtc RNA repair system is linked to virulence in Escherichia coli
Authors: Irakleidi Danai-Athina , Buck Martin , Kotta-Loizou Ioly
Published: May 2022

Antibiotic resistance is one of the biggest public health challenges of the 21st century. The Rtc RNA repair system is present in many pathogenic bacterial species, including the model organism and putative pathogen Escherichia coli, and may play a role in antibiotic resistance. Here we explore its physiological role during infection. Pathogenicity assays are being performed using the infection model Galleria mellonella, to study the phenotypes and survival rates following larvae infection with E. coli K12 and variants lacking rtc genes. Larvae infected with wild-type bacteria survive for up to 10 days as opposed to those lacking any of the rtc genes, which survive for up to 14 days on average. Complementation of the rtc gene deletions with wild-type Rtc proteins, but not functionally catalytic mutants, reverses the infection phenotype to that of wild-type. Similarly, viable larvae infected with wild-type or complemented bacteria score much lower in a health index scoring system than those infected with strains either lacking any of rtc genes or expressing catalytically inactive Rtc proteins. To further explore the importance of Rtc in infection, bacterial burden assays will be conducted to measure the bacterial load. The results so far suggest that bacteria with a fully functional Rtc system are more aggressive in the G. mellonellainfection model. This outcome supports the notion that the Rtc RNA repair system is involved in bacterial virulence and opens the window for further research to understand the ways in which Rtc may contribute to resistance, with potential for broader health benefit.
How do bacteria change their coats? Structural analysis of acyltransferases involved in O-antigen modification
Authors: Tindall Sarah , Woude Marjan van der , Thomas Gavin
Published: May 2022

Acyltransferase-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.
Investigation of the Localisation of FtsZ in Pathogenic E. coli Upon Bacteriophage Addition in a Human Cell Model as a Biomarker for Antibacterial Agents
Authors: Dhanoa Gurneet , Roper David , Sagona Antonia
Published: May 2022

Antimicrobial 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.
Phage display strategy against outbreaks of unknown pathogens
Authors: Palma Marco
Published: May 2022

The experience with SARS-CoV-2 that spread rapidly throughout the world makes us realize we need protocols to act quickly against unknown pathogens. The immune system protects recovering patients from any pathogen by producing antibodies against their immunogenic epitopes. Therefore, the strategy presented here is based on convalescent blood samples and a phage display platform for antibody and peptide drug discovery. Peptide libraries are screened against purified convalescent antibodies to identify immunogenic epitopes of the pathogen. Furthermore, the B cells of the recovery patients are used to amplify variable domains of antibody heavy and light chains expressed during the infection. These domains are cloned in a phagemid and produce free phage particles expressing the antibody fragments on their surfaces to select binders to pathogen immunogenic epitopes. These findings are essential in the identification of the unknown pathogen and the design of therapeutic molecules. In conclusion, this report describes a phage display strategy to combat outbreaks of unknown pathogens such as SARS-CoV-2.
Palmatine and berberine chloride synergistically Inhibit NanH sialidase of Tannerella forsythia
Authors: Galleh P. R. , Lambert D. W. , Stafford G. P.
Published: May 2022

The 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.
A Legionella’s outbreak in a COVID-19 pandemic setting
Authors: Oliveira Catarina , Leite Carla , Rodrigues Helena , Fonseca Fernando
Published: May 2022

In 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.
Bacteriological and Physicochemical Assessment of Sachet and Bottled Drinking Water Sold in Makurdi Metropolis, Benue State, Nigeria
Authors: Agbendeh Lubem , Ogbonna Innocent
Published: May 2022

Background

Packaged 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.

Objective

This study was to assess the bacteriological and physicochemical characteristics of sachet and bottled water sold in Makurdi metropolis.

Methodology

A 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.

Results

The 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.

Conclusion

There 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.